Methods for inhibiting cancer growth

Abstract

The present disclosure provides a method of doing so in a subject in need of treating cancer, the method comprising contacting the cell surface of the subject with an effective amount of a recombinant collagen fragment comprising a fragment having at least about 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. Also provided is a therapeutic biomaterial for use in the methods described herein.

Description

Technical Field

[0001] (Reference to Electronically Filed Sequence Listing) The content of the electronically filed sequence listing XML (name: 4431_088PC01_SequenceListing_ST26.xml, size: 1,312,028 bytes, and creation date: May 30, 2023) filed together with this application is hereby incorporated by reference in its entirety into this specification.

Background Art

[0002] Collagen is one of the most important proteins in the human body and is present in connective tissues such as cartilage, bone, tendon, ligament, and skin. Collagen is the major protein in the extracellular matrix of human cells. "Recombinant collagen" refers to a family of at least 28 distinct naturally occurring collagen types prepared using recombinant techniques.

[0003] Despite recent advances in cancer therapy, cancer remains a leading cause of death worldwide, with nearly 10 million deaths in 2020. One defining feature of cancer is the rapid generation of abnormal cells that can invade and seed distant organs far from the primary tumor or neoplasm. These metastatic cells may remain dormant for years before forming clinically detectable metastatic tumors. Widespread metastasis is the main cause of death from cancer. Cancer treatment modalities such as immunotherapy are a common area of clinical research, but there remains a need in the field of oncology for creative approaches and novel therapies to treat the primary tumor and prevent metastatic seed cells from becoming incurable metastatic tumors.

Summary of the Invention

[0004] The present disclosure provides novel and inventive methods and compositions for performing it in a subject in need of inhibiting cancer growth. In some embodiments, the present disclosure is a method for performing it in a subject in need of treating cancer, the method comprising contacting the cell surface of the subject with an effective amount of a recombinant collagen fragment having at least about 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the recombinant collagen fragment is not hydroxylated. In some embodiments, the recombinant collagen fragment is hydroxylated.

[0005] In some embodiments, the collagen fragment has the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the collagen fragment having the amino acid sequence set forth in SEQ ID NO: 1 is not hydroxylated. In some embodiments, the collagen fragment having the amino acid sequence set forth in SEQ ID NO: 1 is hydroxylated.

[0006] In some embodiments, the present disclosure is a method for performing it in a subject in need of treating cancer, the method comprising contacting the cell surface of the subject with an effective amount of a recombinant collagen fragment having the amino acid sequence set forth in any one of SEQ ID NOs: 2-972.

[0007] In some embodiments, inducing cell dormancy by contacting the cell surface with the fragment.

[0008] In some embodiments, the cancer is characterized by the presence of a solid tumor. In some embodiments, the cancer is skin, colon, oral mucosa, rectum, esophagus, thyroid, liver, pancreas, kidney, bladder, lung, brain, breast, ovary, testis, prostate, bone cancer, or any combination of the foregoing.

[0009] In some embodiments, the subject has not undergone tumor resection. In some embodiments, the subject has undergone resection of the primary tumor. In some embodiments, the cell surface of the subject contains remaining cancer cells after resection of the primary tumor.

[0010] In some embodiments, cell dormancy is maintained by contacting the cell surface with a fragment.

[0011] In some embodiments, the present disclosure provides a method of inhibiting cancer cell growth, the method comprising contacting a cancer cell with a recombinant collagen fragment having at least about 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the recombinant collagen fragment is not hydroxylated. In some embodiments, the recombinant collagen fragment is hydroxylated.

[0012] In some embodiments of the method of inhibiting cancer cell growth disclosed herein, the collagen fragment has the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the recombinant collagen fragment is not hydroxylated. In some embodiments, the recombinant collagen fragment is hydroxylated.

[0013] In some embodiments, the present disclosure provides a method of inhibiting cancer cell growth, the method comprising contacting a cancer cell with a recombinant collagen fragment having the amino acid sequence set forth in any one of SEQ ID NOs: 2-972.

[0014] In some embodiments of the methods disclosed herein, the collagen fragment is formulated in a pharmaceutically acceptable composition. In some embodiments, the fragment is formulated in a therapeutic biomaterial.

[0015] In some embodiments, the polydispersity of the collagen fragments provided herein (the weight-average molecular weight of the collagen fragments divided by the number-average molecular weight of the collagen fragments) can be about 1. In some embodiments, the polydispersity of the collagen fragments can be in the range of about 1 to about 2. In some embodiments, the polydispersity can be in the range of about 1 to about 3 or about 1 to about 5.

[0016] In some embodiments, the therapeutic biomaterial can be a protein-polyurethane alloy. In some embodiments, the protein is dissolved in the polyurethane. In some embodiments, when the alloy is exposed to water, the protein moves from the alloy and contacts the cell surface. In some embodiments, when the alloy is exposed to a buffer solution, the protein moves from the alloy and contacts the cell surface. In some embodiments, the alloy can include from about 10 wt% to about 50 wt% of the fragments and from about 50 wt% to about 90 wt% of the polyurethane. In some embodiments, the alloy can include from about 20 wt% to about 35 wt% of the protein and from about 65 wt% to about 80 wt% of the polyurethane. In some embodiments, the alloy may or may substantially not contain particles of recombinant collagen fragments having an average diameter greater than 1 micron.

[0017] In some embodiments, the present disclosure provides a therapeutic biomaterial comprising a recombinant collagen fragment having at least about 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 and a pharmaceutically acceptable scaffold. In some embodiments, the recombinant collagen fragment within the pharmaceutically acceptable scaffold may not be hydroxylated. In some embodiments, the recombinant collagen fragment within the pharmaceutically acceptable scaffold may be hydroxylated.

[0018] In some embodiments, the recombinant collagen fragment within a pharmaceutically acceptable scaffold has the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the recombinant collagen fragment within a pharmaceutically acceptable scaffold may not be hydroxylated. In some embodiments, the recombinant collagen fragment within a pharmaceutically acceptable scaffold may be hydroxylated.

[0019] In some embodiments, the present disclosure provides a therapeutic biomaterial comprising a recombinant collagen fragment having a sequence according to any one of SEQ ID NOs: 2 to 972 and a pharmaceutically acceptable scaffold.

[0020] In some embodiments, the present disclosure provides a therapeutic biomaterial comprising a recombinant collagen fragment having at least about 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 for use in treatment in a subject in need of treatment for cancer and a pharmaceutically acceptable scaffold. In some embodiments, the cancer may be characterized by the presence of a solid tumor. In some embodiments, the cancer can be skin, colon, oral mucosa, rectum, esophagus, thyroid, liver, pancreas, kidney, bladder, lung, brain, breast, ovary, testis, prostate, bone cancer, or any combination of the foregoing.

[0021] In some embodiments, the pharmaceutically acceptable scaffold can be a protein-polyurethane alloy, and the protein is dissolved in the polyurethane. The protein-polyurethane alloy is described, for example, in U.S. Patent No. 2021-0355326, which is incorporated herein by reference in its entirety. In some embodiments, when the alloy is exposed to water or a buffer solution, the protein moves from the alloy and contacts the cell surface. In some embodiments, when the alloy is exposed to the cell surface, the protein moves from the alloy.

[0022] In some embodiments, the alloy comprises from about 10 wt% to about 50 wt% of the fragment and from about 50 wt% to about 90 wt% of the polyurethane. In some embodiments, the alloy comprises from about 20 wt% to about 35 wt% of the protein and from about 65 wt% to about 80 wt% of the polyurethane. In some embodiments, the alloy does not contain or substantially does not contain particles of recombinant collagen fragments having an average diameter greater than 1 micron.

[0023] In some embodiments, the pharmaceutically acceptable scaffold comprises a protein hyaluronic acid alloy. In some embodiments, the hyaluronic acid and the protein are crosslinked by themselves, with each other, or both. In some embodiments, the protein is dissolved in the crosslinked hyaluronic acid matrix. In some embodiments, when the alloy is exposed to water, a buffer solution, and / or a cell surface, the protein moves from the alloy.

[0024] In some embodiments, the pharmaceutically acceptable scaffold comprises a protein dissolved in a gelatin matrix. In some embodiments, the gelatin is crosslinked. In some embodiments, when the alloy is exposed to water or a buffer solution, the protein moves from the alloy and contacts the cell surface.

[0025] In some embodiments, the biomaterial adheres directly onto the cancer cells.

[0026] In some embodiments, the present disclosure provides the use of the biomaterials described herein for treating a wound resulting from resection of a primary tumor.

[0027] In some embodiments, the present disclosure provides the use of the biomaterials described herein for maintaining cell quiescence.

[0028] In some embodiments, the present disclosure provides a therapeutic biomaterial comprising a recombinant collagen fragment having at least about 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 and a pharmaceutically acceptable scaffold. In some embodiments, the recombinant collagen fragment is not hydroxylated. In some embodiments, the recombinant collagen fragment is hydroxylated.

[0029] In some embodiments, the collagen fragment has the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the recombinant collagen fragment is not hydroxylated. In some embodiments, the recombinant collagen fragment is hydroxylated.

[0030] In some embodiments, the present disclosure provides a therapeutic biomaterial comprising a recombinant collagen fragment having a sequence comprising the amino acid sequence set forth in any one of SEQ ID NOs: 2 to 972 or the amino acid sequence set forth in any one of SEQ ID NOs: 974 to 1002 and a pharmaceutically acceptable scaffold.

[0031] In some embodiments, the present disclosure provides a therapeutic biomaterial for use in the treatment of a subject in need thereof for the treatment of cancer, comprising a recombinant collagen fragment having at least about 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 and a pharmaceutically acceptable scaffold. In some embodiments, the cancer is characterized by the presence of a solid tumor. In some embodiments, the cancer is skin, colon, oral mucosa, rectum, esophagus, thyroid, liver, pancreas, kidney, bladder, lung, brain, breast, ovary, testis, prostate, bone cancer, or any combination of the foregoing.

[0032] In some embodiments, the present disclosure provides a biomaterial comprising a pharmaceutically acceptable scaffold. In some embodiments, the pharmaceutically acceptable scaffold comprises a mixture of hyaluronic acid and recombinant collagen fragments. In some embodiments, the recombinant collagen fragments are mixed with hyaluronic acid. In some embodiments, the hyaluronic acid scaffold is crosslinked. In some embodiments, the hyaluronic acid is a biphasic crosslinked hyaluronic acid comprising crosslinked HA and non-crosslinked HA.

[0033] In some embodiments, the present disclosure provides a biomaterial comprising a pharmaceutically acceptable scaffold, wherein the pharmaceutically acceptable scaffold comprises a mixture of polyvinylpyrrolidone and recombinant collagen fragments.

[0034] In some embodiments, the pharmaceutically acceptable scaffold comprises a mixture of polyacrylamide and recombinant collagen fragments.

[0035] In some embodiments, the pharmaceutically acceptable scaffold comprises a mixture of poly(ethylene oxide) and recombinant collagen fragments.

[0036] In some embodiments, the pharmaceutically acceptable scaffold comprises a mixture of poly(2-oxazoline) and recombinant collagen fragments.

[0037] In some embodiments, the pharmaceutically acceptable scaffold comprises a mixture of polyethyleneimine and recombinant collagen fragments.

[0038] In some embodiments, the pharmaceutically acceptable scaffold comprises a mixture of carboxymethylcellulose and recombinant collagen fragments.

[0039] In some embodiments, the pharmaceutically acceptable scaffold comprises a mixture of chondroitin sulfate and recombinant collagen fragments.

[0040] In some embodiments, the pharmaceutically acceptable scaffold comprises a mixture of acetylated hyaluronic acid and recombinant collagen fragments.

[0041] In some embodiments, the pharmaceutically acceptable scaffold comprises a mixture of zinc hyaluronate and recombinant collagen fragments.

[0042] In some embodiments, the biomaterial directly adheres onto cancer cells.

[0043] In some embodiments, the present disclosure provides the use of the biomaterial of the present disclosure for treating a wound resulting from resection of a primary tumor.

[0044] In some embodiments, the present disclosure provides uses for maintaining cell quiescence.

[0045] In some embodiments, the present disclosure provides a method of doing so in a subject in need of treating cancer, the method comprising contacting the cell surface of the subject with a therapeutically effective amount of a biomaterial comprising a recombinant collagen fragment having at least about 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the recombinant collagen is not hydroxylated. In some embodiments, the recombinant collagen is hydroxylated.

[0046] In some embodiments, the present disclosure provides a method of inhibiting the growth of cancer cells, the method comprising contacting the cancer cells with a therapeutically effective amount of a biomaterial comprising a recombinant collagen fragment having the amino acid sequence set forth in SEQ ID NO: 973. In some embodiments, the recombinant collagen is not hydroxylated. In some embodiments, the recombinant collagen is hydroxylated.

[0047] In some embodiments, the present disclosure provides a method of doing so in a subject in need of inducing cell dormancy, the method comprising contacting the cell surface in the subject with a therapeutic biomaterial comprising recombinant collagen having the amino acid sequence set forth in SEQ ID NO: 973. In some embodiments, the recombinant collagen is not hydroxylated. In some embodiments, the recombinant collagen is hydroxylated.

[0048] In some embodiments of the methods of treating cancer provided herein, the cancer is characterized by the presence of a solid tumor. In some embodiments, the cancer is skin, colon, oral mucosa, rectal, esophageal, thyroid, liver, pancreatic, kidney, bladder, lung, brain, breast, ovarian, testicular, prostate, bone cancer, or any combination of the foregoing.

[0049] In some embodiments of the methods provided herein, the therapeutic biomaterial is a protein polyurethane alloy.

[0050] In some embodiments of the methods provided herein, the therapeutic biomaterial comprises polyurethane, hyaluronic acid, or gelatin.

[0051] In some embodiments of the methods provided herein, the therapeutic biomaterial comprises polyvinylpyrrolidone.

[0052] In some embodiments of the methods provided herein, the therapeutic biomaterial comprises polyacrylamide.

[0053] In some embodiments of the methods provided herein, the therapeutic biomaterial comprises poly(ethylene oxide).

[0054] In some embodiments of the methods provided herein, the therapeutic biomaterial comprises poly(2-oxazoline).

[0055] In some embodiments of the methods provided herein, the therapeutic biomaterial comprises polyethyleneimine.

[0056] In some embodiments of the methods provided herein, the therapeutic biomaterial comprises carboxymethylcellulose.

[0057] In some embodiments of the methods provided herein, the therapeutic biomaterial comprises chondroitin sulfate.

[0058] In some embodiments of the methods provided herein, the therapeutic biomaterial comprises acetylated hyaluronic acid.

[0059] The method according to any one of claims 60 to 66, wherein the therapeutic biomaterial comprises acetylated hyaluronic acid.

[0060] In some embodiments of the methods provided herein, the therapeutic biomaterial comprises zinc hyaluronate.

[0061] In some embodiments of the methods provided herein, the therapeutic biomaterial adheres directly to cancer cells.

[0062] In some embodiments of the methods provided herein, the polydispersity of the collagen fragments is 1 to 2, 1 to 3, or 1 to 5. In some embodiments, the polydispersity of the collagen fragments is 1 to 2, 1 to 3, or 1 to 5.

Brief Description of the Drawings

[0063]

Figure 1A

Figure 1B

Figure 2A

Figure 2B

Figure 2C

Figure 2D

Figure 2E

Figure 2F

Figure 2G

Figure 2H

Figure 2I

Figure 2J

Figure 3

Figure 4

Figure 5A

Figure 5B

Figure 5C

Figure 5D

Figure 5E

Figure 6A

Figure 6B

Figure 6C

Figure 6D

Figure 6E

Figure 6F

Figure 6G

Figure 6H

Figure 6I

Figure 6J

Figure 7

Figure 8

Figure 9A

Figure 9B

Figure 9C

Figure 9D

Figure 9E

Figure 9F

Figure 10A

Figure 10B

Figure 10C

Figure 10D

Figure 10E

Figure 11A

Figure 11B

Figure 12A

Figure 12B

Figure 13A

Figure 13B

Figure 14A

Figure 14B

DETAILED DESCRIPTION OF THE INVENTION

[0064] Definitions The indefinite articles "a" and "an" used to describe an element or component mean that one or at least one of these elements or components is present. These articles, when used in this specification, are conventionally used to indicate that the modified noun is a singular noun, but unless specifically stated otherwise in a particular example, the articles "a" and "an" also include the plural form. Similarly, when used in this specification, the definite article "the" also means that the modified noun can be either singular or plural, again unless specifically stated otherwise in a particular example.

[0065] As used herein, the term "about" when used with a numerical value means within 10% of the recited value, unless otherwise clearly stated. For example, "about 5 wt%" means 4.5 wt% to 5.5 wt%. Notwithstanding the foregoing, "about" should not be understood to modify the value to more than 100%. For example, a composition containing "about 95" weight percent of a given element can have from 85.5 to 99.999 weight percent of the component in the composition.

[0066] The term "collagen" refers to any one of the known collagen types, whether natural, synthetic, semi-synthetic, or recombinant. The term "collagen" includes collagen, fragments of collagen, collagen-like proteins, triple helix collagen, alpha chains, monomers, gelatin, trimers, and combinations thereof. It includes all of the collagen, modified collagen, and collagen-like proteins described herein. The term also encompasses procollagen and collagen-like or collagenous proteins that include the motif (Gly-X-Y)n, where n is an integer. It includes molecules of collagen and collagen-like proteins, trimers of collagen molecules, fibrils of collagen, and fibers of collagen fibrils. It also refers to chemically, enzymatically, or recombinantly modified collagen or collagen-like molecules that can fibrillate, and fragments of collagen, collagen-like molecules, and collagenous molecules that can be assembled into nanofibers. Recombinant collagen molecules, whether natural or engineered, generally contain a repeated -(Gly-X-Y)n sequence.

[0067] As used herein, collagen is a general term for a family of at least 28 distinct collagen types. A variety of distinct collagen types have been identified in a wide range of species, including bovine, ovine, porcine, avian, marine, plant, and human collagen. Animal skin is typically type I collagen. The term "collagen" encompasses unprocessed (e.g., procollagen), post-translationally modified collagen having a triple helix structure, and proteolyzed collagen. Type I collagen is the major fibrillar collagen of bone and skin, constituting approximately 80-90% of the total collagen in an organism. Type I collagen is a major structural macromolecule present in the extracellular matrix of multicellular organisms and comprises approximately 20% of the total protein mass. Type I collagen is a heterotrimeric molecule containing two α1(I) chains and one α2(I) chain, each encoded by the COL1A1 and COL1A2 genes, respectively. The in vivo assembly of type I collagen fibrils, fibers, and fiber bundles occurs during growth and provides mechanical support to tissues while enabling cell motility and nutrient transport. Other collagen types are less abundant than type I collagen and exhibit different distribution patterns. Type III collagen is the major fibrillar collagen found in skin and vascular tissue. Type III collagen is a homotrimeric collagen containing three identical α1(III) chains encoded by the COL3A1 gene.

[0068] The term "modified" as applied to the collagen fragments disclosed herein refers to a collagen fragment comprising an amino acid sequence that is at least 70%, 80%, 90%, 95%, or 99% identical or similar to the amino acid sequence of a biologically active molecule. In some embodiments, the modified collagen fragment comprises an amino acid sequence that is at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of a native sequence or a previously engineered sequence. The modified sequence may include additions, deletions, substitutions, or combinations thereof to the amino acid sequence of a native sequence or a previously engineered molecule. For example, a modified collagen fragment may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid residues incorporated or deleted compared to the native collagen sequence. Such selections may be made to modify the looseness or tension of recombinant collagen. The degree of hydroxylation of collagen correlates with the looseness or tension of the collagen triple helix. Modified collagen fragments may also include chemical modifications to the polypeptide, such as cross-linking between cysteine residues, or between hydroxylated or glycosylated residues, or hydrolysis to a low molecular weight by any mechanism.

[0069] The term "pharmaceutically acceptable" as applied to a carrier, excipient, or stabilizer that can be used in the compositions described herein refers to a carrier, excipient, or stabilizer that is non-toxic to the recipient at the dosage and concentration used.

[0070] As used herein, the term "protein" refers to any of the collagen fragments described herein, including 1) "50 kDa rCol", 2) recombinant collagen fragments having at least about 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, 3) recombinant collagen fragments having at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity or similarity to SEQ ID NO: 1, and 4) any combination of the foregoing.

[0071] As used herein, the terms "hyaluronic acid" and "hyaluronic acid, HA" refer to acidic polysaccharides composed of residues of D-glucuronic acid and N-acetyl-D-glucosamine, having different molecular weights, and their corresponding salts including, but not limited to, sodium hyaluronate. As used herein, a composition, scaffold, therapeutic biomaterial, or any contemplated embodiment containing HA is to be understood to include a combination of hyaluronic acid and sodium hyaluronate, unless otherwise specified.

[0072] As used herein, the terms "sodium carboxymethylcellulose", "carboxymethylcellulose", and "carboxymethylcellulose, CMC" may be used interchangeably and refer to polymers having different ranges of molecular weights as derivatives of cellulose in which a carboxymethyl group is attached to a part of the monomer constituting the cellulose backbone. Compositions described herein as containing carboxymethylcellulose are to be understood to include a combination of carboxymethylcellulose and sodium carboxymethylcellulose, unless otherwise specified.

[0073] As used herein, the term "miscibility" means that one substance is soluble in the other such that when two components are mixed together in water or a buffer system, a molecularly homogeneous mixture is formed. In solution, collagen that is completely miscible with hyaluronic acid refers to a solution that is a uniformly mixed or blended combination that forms a transparent single phase under suitable process conditions and in which no visually detectable collagen particles are present. Solid films prepared from completely miscible solutions may appear visually transparent, may appear cloudy, and / or may exhibit phase separation. Transparency can be readily determined by one of ordinary skill in the art and can be measured, for example, using a nephelometer.

[0074] In the context of a protein-polyurethane alloy, a protein that is miscible with a polyurethane means that the protein can be miscible with only one of the plurality of phases of the polyurethane with which it is blended or with only one of a plurality of polyurethanes.

[0075] A. Method of treating cancer In some embodiments, the present disclosure provides a method of doing so in a subject in need of treating cancer, the method comprising contacting the cell surface of the subject with an effective amount of a recombinant collagen fragment.

[0076] As used herein, the term "recombinant collagen" refers to a family of at least 28 distinct naturally-occurring collagen types, including, but not limited to, types I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV, XV, XVI, XVII, XVIII, XIX, and XX, prepared using recombinant techniques. The term collagen includes collagen, collagen fragments, collagen-like proteins, triple-helical collagen, alpha chains, monomers, gelatin, trimers, and combinations thereof. Recombinant expression of collagen and collagen-like proteins is known in the art (see, e.g., Bell, European Patent No. 1232182 (B1), Bovine collagen and method for producing recombinant gelatin, Olsen, et al., U.S. Patent No. 6,428,978, and VanHeerde, et al., U.S. Patent No. 8,188,230, which are hereby incorporated by reference in their entirety). Collagen is characterized by an amino acid repeating triplet, -(Gly-X-Y)n-, such that approximately one-third of the amino acid residues in collagen are glycine. X is often proline and Y is often hydroxyproline. The structure of collagen may consist of three intertwined peptide chains of different lengths.

[0077] In some embodiments, the recombinant collagen described herein is a recombinant collagen fragment. The recombinant collagen fragment may be a fragment of the complete amino acid sequence of a native collagen molecule that can form tropocollagen (trimeric collagen), or the fragment may be a modified collagen molecule or a fragment of a truncated collagen molecule having an amino acid sequence that is at least 70, 80, 90, 95, 96, 97, 98, or 99% identical or similar to the native collagen amino acid sequence (or to its fibril-forming region, or to a segment substantially containing [Gly-X-Y]n).

[0078] Exemplary collagen sequences from which the fragments can be derived include those described by accession number P02461.4 (SEQ ID NO: 1003; human Col3A1) (www.ncbi.nlm.nih.gov / protein / 124056490), P02452 (SEQ ID NO: 982; human Col1A1), P08123 (SEQ ID NO: 983; human Col1A2), NP_001029211.1 (SEQ ID NO: 1004; bovine Col1A1) (www.ncbi.nlm.nih.gov / protein / 77404252), NP_776945.1 (SEQ ID NO: 1005; bovine Col1A2) (www.ncbi.nlm.nih.gov / protein / 27806257), and NP_001070299.1 (SEQ ID NO: 1006; bovine (Col3A1) (www.ncbi.nlm.nih.gov / protein / 116003881), including the amino acid sequences of Col1A1, Col1A2, and Col3A1, which are incorporated herein by reference.

[0079] In some embodiments, the collagen fragment has at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the collagen fragment has the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the collagen fragment has the amino acid sequence set forth in any one of SEQ IDs NO: 2 - 972.

[0080] In some embodiments, the collagen fragment can be a collagen fragment sequence variant having the amino acid sequence by any one of SEQ IDs NO: 975 - 1002.

[0081] In some embodiments, the present disclosure provides a method of doing so in a subject in need of treating cancer, the method comprising contacting the cell surface of the subject with an effective amount of a recombinant collagen fragment having at least about 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. SEQ ID NO: 1 has a molecular weight of about 50 kDa. In some embodiments, the recombinant collagen fragment can have at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity or similarity to SEQ ID NO: 1.

[0082] The amino acid sequence of SEQ ID NO: 1 is as follows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

[0083] In some embodiments, lysine, proline, or lysine and proline residues present in the recombinant collagen fragment are not hydroxylated. In other embodiments, the recombinant collagen fragments described herein may be hydroxylated. In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% (or any intermediate value or subrange) of the lysine, proline, or lysine and proline residues in the recombinant collagen fragment may be hydroxylated. Hydroxylating collagen can build rheology and improve the thermal stability of collagen molecules or fragments. Hydroxylated collagen and hydroxylated collagen fragments are also resistant, for example, to high-concentration pepsin digestion at a pepsin:total protein ratio of 1:25 to 1:1. In some embodiments, the recombinant collagen fragment having the amino acid sequence set forth in SEQ ID NO: 1 is not hydroxylated. In some embodiments, the recombinant collagen fragment having the amino acid sequence set forth in SEQ ID NO: 1 is hydroxylated.

[0084] In some embodiments, the present disclosure provides a method of doing so in a subject in need of treating cancer, the method comprising contacting the cell surface of the subject with an effective amount of a recombinant collagen fragment having the amino acid sequence set forth in any one of SEQ ID NOs: 2-972. In some embodiments, the recombinant collagen may comprise a hydrolysis product of the collagen fragment, and the hydrolysis product may have a sequence that is a portion of SEQ ID NO: 1. In some embodiments, the hydrolysis product may have a sequence according to one of SEQ ID NOs: 2-972.

[0085] In some embodiments, the methods provided herein of doing so in a subject in need of treating cancer do not include contacting the cell surface of the subject with an effective amount of a recombinant collagen having the amino acid sequence set forth in SEQ ID NO: 973 in the absence of a pharmaceutically acceptable scaffold.

[0086] In some embodiments, the recombinant collagen fragments described herein may have the amino acid sequences set forth in Table 1 below.

[0087]

Table 1-1

[0088]

Table 1-2

[0089]

Table 1-3

[0090]

Table 1-4

[0091]

Table 1-5

[0092]

Table 1-6

[0093]

Table 1-7

[0094]

Table 1-8

[0095]

Table 1-9

[0096]

Table 1-10

[0097]

Table 1-11

[0098]

Table 1-12

[0099]

Table 1-13

[0100]

Table 1-14

[0101]

Table 1-15

[0102]

Table 1-16

[0103]

Table 1-17

[0104]

Table 1-18

[0105]

Table 1-19

[0106]

Table 1-20

[0107]

Table 1-21

[0108]

Table 1-22

[0109]

Table 1-23

[0110]

Table 1-24

[0111]

Table 1-25

[0112] As described above in this specification, it is well known in the art that cancer cells may remain dormant for years before spreading to distal organs, seeding, and forming clinically detectable metastases. In addition, disseminated tumor cells may sense and remodel the extracellular matrix (ECM) to sustain dormancy. Dormant cancer cells construct an ECM niche rich in type III collagen. Without wishing to be bound by any particular theory, it is thought that the enrichment of type III collagen in the ECM can limit or prevent metastasis through induction of dormancy in disseminated tumor cells.

[0113] In some embodiments of the methods of treating cancer disclosed herein, cell dormancy is induced by contacting the cell surface in a subject having cancer with a recombinant collagen fragment. In some embodiments, the cancer is characterized by the presence of a solid tumor. In some embodiments, the cancer can be cancer of the skin, colon, oral mucosa, rectum, esophagus, thyroid, liver, pancreas, kidney, bladder, lung, brain, breast, ovary, testis, prostate, bone, or any combination of the foregoing.

[0114] In some embodiments, the subject has not undergone tumor resection. In some embodiments, the subject has undergone resection of the primary tumor. In some embodiments, the cell surface of the subject contains cancer cells remaining after resection of the primary tumor.

[0115] In some embodiments of the methods of treating cancer disclosed herein, cell dormancy is maintained by contacting the cell surface in a subject having cancer with a fragment.

[0116] In some embodiments, the present disclosure provides a method of inhibiting the growth of cancer cells, the method comprising contacting the cancer cells with a recombinant collagen fragment having at least about 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 and / or 973. When this specification uses the phrase "1 and / or 973", it is understood that any percent identity preceding this phrase means that the intended collagen sequence can have the described percent identity to either sequence individually or to both sequences (i.e., as a mixture). In some embodiments, the collagen fragment can have at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity or similarity to SEQ ID NO: 1 and / or 973. In some embodiments, the present disclosure provides a method of inhibiting the growth of cancer cells, the method comprising contacting the cancer cells with a recombinant collagen fragment having at least about 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 and / or 973. In some embodiments, the collagen fragment can have at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity or similarity to SEQ ID NO: 1, and the recombinant collagen fragment has the amino acid sequence set forth in any one of SEQ ID NOs: 974 - 1002.

[0117] In some embodiments, the recombinant collagen fragment may not be hydroxylated. In some embodiments, the recombinant collagen fragment may be hydroxylated.

[0118] In some embodiments, the present disclosure provides a method of inhibiting the growth of cancer cells, the method comprising contacting the cancer cells with a recombinant collagen fragment having an amino acid sequence set forth in any one of SEQ ID NOs: 2 to 972.

[0119] In some embodiments, the method of inhibiting the growth of cancer cells provided herein does not include contacting the cancer cells with a recombinant collagen having the amino acid sequence set forth in SEQ ID NO: 973 in the absence of a pharmaceutically acceptable scaffold.

[0120] In some embodiments, the fragment is formulated in a pharmaceutically acceptable composition. In some embodiments, the pharmaceutically acceptable composition includes at least one pharmaceutically acceptable excipient. In certain embodiments, the composition may include a recombinant collagen fragment according to SEQ ID NO: 1 and / or 973 and at least one pharmaceutically acceptable excipient. In still further embodiments, the composition may include a recombinant collagen fragment having about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identity or similarity to SEQ ID NO: 1 or SEQ ID NO: 973, wherein the recombinant collagen fragment has an amino acid sequence set forth in any one of SEQ ID NOs: 974 to 1002, and at least one pharmaceutically acceptable excipient. In some embodiments, the composition may include a recombinant collagen fragment having an amino acid sequence of any one of SEQ ID NOs: 2 to 972 and at least one pharmaceutically acceptable excipient.

[0121] In another embodiment, the composition may comprise a recombinant collagen fragment according to SEQ ID NO: 1 and / or 973, a plurality of hydrolyzates having such sequences that may be the same or different according to any one of SEQ ID NOs: 2 to 972, and at least one pharmaceutically acceptable excipient. In certain embodiments, the number of hydrolyzates present in the plurality of hydrolyzates in the composition may increase over time, with temperature, with pH, or as a result of other conditions that typically cause hydrolysis or degradation of a recombinant collagen fragment such as the recombinant collagen fragment according to SEQ ID NO: 1 or 973. In other embodiments, the composition may be stabilized with one or more stabilizers such that the concentration of the recombinant collagen fragment according to SEQ ID NO: 1 and / or 973, as well as the concentration of each of the fragments in the plurality of fragments, remains substantially constant (i.e., varies by no more than ±5% by HPLC over a given period) or remains constant. In certain embodiments, a recombinant collagen fragment such as the recombinant collagen fragment according to SEQ ID NO: 1 may be hydrolyzed such that less than about 10%, about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, or about 90% to about 100% of the non-hydrolyzed recombinant fragment remains in the composition as measured by HPLC. In other embodiments, the composition may comprise a mixture of a recombinant collagen fragment (e.g., a recombinant collagen fragment according to SEQ ID NO: 1) and a plurality of hydrolyzates of the recombinant collagen fragment (e.g., a plurality of collagen fragments according to any of SEQ ID NOs: 2 to 972) such that the weight of the hydrolyzates in the composition is less than about 10%, about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, or about 90% to about 100% of the weight of the collagen-related proteins in the composition.

[0122] The pharmaceutically acceptable compositions for use in the methods disclosed herein have the desired degree of purity in a physiologically acceptable carrier, excipient, or stabilizer (Remington’s Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA). Acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the dosages and concentrations employed and include buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (octadecyl dimethyl benzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzetonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol, etc.); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as TWEEN (registered trademark), PLURONICS (registered trademark), or polyethylene glycol (PEG).

[0123] In some embodiments, a topical mixture comprising a recombinant collagen fragment can be prepared for topical administration. The resulting mixture can be a solution, suspension, emulsion, etc., and can be formulated as a cream, gel, ointment, emulsion, solution, elixir, lotion, suspension, tincture, paste, foam, aerosol, irrigation, spray, bandage, skin patch, or any other formulation suitable for topical administration. The topical mixtures described herein can be formulated for topical application to the skin and mucosa in the form of gels, creams, and lotions for transdermal delivery.

[0124] Transdermal patches containing iontophoresis devices and electrophoresis devices are well known to those skilled in the art and can be used to administer recombinant collagen fragments. For example, such patches are disclosed in U.S. Patent Nos. 6,267,983, 6,261,595, 6,256,533, 6,167,301, 6,024,975, 6,010715, 5,985,317, 5,983,134, 5,948,433, and 5,860,957, each of which is incorporated herein by reference in its entirety.

[0125] B. Therapeutic Biomaterials In some embodiments of the methods disclosed herein, the present disclosure provides a therapeutic biomaterial comprising a recombinant collagen fragment having at least about 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 and a pharmaceutically acceptable scaffold. In some embodiments of the methods disclosed herein, the present disclosure provides a therapeutic biomaterial comprising a recombinant collagen fragment having a sequence comprising the amino acid sequence set forth in any one of SEQ ID NOs: 2 - 972 and a pharmaceutically acceptable scaffold. In some embodiments of the methods disclosed herein, the present disclosure provides a therapeutic biomaterial comprising a recombinant collagen fragment having at least about 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, wherein the recombinant collagen fragment has the amino acid sequence set forth in any one of SEQ ID NOs: 974 - 1002 and a pharmaceutically acceptable scaffold.

[0126] As used herein, a pharmaceutically acceptable scaffold can include a synthetic, polymeric, non-immunogenic, three-dimensional scaffold. Suitable scaffolds can be biocompatible and biodegradable support structures and can be gel scaffolds such as solid, liquid, or injectable gel scaffolds. Suitable scaffolds can also include those made from nanomaterials such as nanopolymers and nanofibers. Such scaffolds can be placed on the cell surface in a subject, e.g., a human subject, following damage to the cell surface or surgery. Suitable scaffolds include any conventionally used and / or available scaffolds for surgery, wound healing, and / or tissue repair and / or manipulation. To create a biocompatible and biodegradable scaffold, some high-performance thermoresponsive polymers such as chitosan, polyvinylpyrrolidone, alginate, and poly(ε-caprolactone) can be used. These processed thermoresponsive biomaterials have a 3D architecture similar to human structures and can be used as an active agent delivery system.

[0127] Additional polymers suitable for use in the pharmaceutically acceptable scaffolds described herein include, but are not limited to, biphasic cross-linked hyaluronic acid including cross-linked HA and non-cross-linked HA, polyvinylpyrrolidone, polyacrylamide, poly(ethylene oxide), poly(2-oxazoline) and protein, polyethyleneimine and protein, carboxymethylcellulose, chondroitin sulfate, acetylated hyaluronic acid, and zinc hyaluronate.

[0128] Thus, in some embodiments of the methods disclosed herein, the present disclosure provides a therapeutic biomaterial comprising a recombinant collagen fragment having at least about 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, and a biphasic crosslinked hyaluronic acid comprising crosslinked HA and non-crosslinked HA. In some embodiments of the methods disclosed herein, the present disclosure provides a therapeutic biomaterial comprising a recombinant collagen fragment having a sequence comprising the amino acid sequence set forth in any one of SEQ ID NOs: 2-972, and a biphasic crosslinked hyaluronic acid comprising crosslinked HA and non-crosslinked HA. In some embodiments of the methods disclosed herein, the present disclosure provides a therapeutic biomaterial comprising a recombinant collagen fragment having at least about 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, wherein the recombinant collagen fragment has the amino acid sequence set forth in any one of SEQ ID NOs: 974-1002, and a biphasic crosslinked hyaluronic acid comprising crosslinked HA and non-crosslinked HA.

[0129] Thus, in some embodiments of the methods disclosed herein, the present disclosure provides a therapeutic biomaterial comprising a recombinant collagen fragment having at least about 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, and polyvinylpyrrolidone. In some embodiments of the methods disclosed herein, the present disclosure provides a therapeutic biomaterial comprising a recombinant collagen fragment having a sequence comprising the amino acid sequence set forth in any one of SEQ ID NOs: 2-972, and polyvinylpyrrolidone. In some embodiments of the methods disclosed herein, the present disclosure provides a therapeutic biomaterial comprising a recombinant collagen fragment having at least about 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, wherein the recombinant collagen fragment has the amino acid sequence set forth in any one of SEQ ID NOs: 974-1002, and polyvinylpyrrolidone.

[0130] In some embodiments of the methods disclosed herein, the present disclosure provides a therapeutic biomaterial comprising a recombinant collagen fragment having at least about 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 and polyacrylamide. In some embodiments of the methods disclosed herein, the present disclosure provides a therapeutic biomaterial comprising a recombinant collagen fragment having a sequence comprising any one of the amino acid sequences set forth in SEQ ID NOs: 2 to 972 and polyacrylamide. In some embodiments of the methods disclosed herein, the present disclosure provides a therapeutic biomaterial comprising a recombinant collagen fragment having at least about 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, wherein the recombinant collagen fragment has an amino acid sequence set forth in any one of SEQ ID NOs: 974 to 1002, and polyacrylamide.

[0131] In some embodiments of the methods disclosed herein, the present disclosure provides a therapeutic biomaterial comprising a recombinant collagen fragment having at least about 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 and poly(ethylene oxide). In some embodiments of the methods disclosed herein, the present disclosure provides a therapeutic biomaterial comprising a recombinant collagen fragment having a sequence comprising any one of the amino acid sequences set forth in SEQ ID NOs: 2 to 972 and poly(ethylene oxide). In some embodiments of the methods disclosed herein, the present disclosure provides a therapeutic biomaterial comprising a recombinant collagen fragment having at least about 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, wherein the recombinant collagen fragment has an amino acid sequence set forth in any one of SEQ ID NOs: 974 to 1002, and poly(ethylene oxide).

[0132] In some embodiments of the methods disclosed herein, the present disclosure provides a therapeutic biomaterial comprising a recombinant collagen fragment having at least about 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 and poly(2-oxazoline). In some embodiments of the methods disclosed herein, the present disclosure provides a therapeutic biomaterial comprising a recombinant collagen fragment having a sequence comprising the amino acid sequence set forth in any one of SEQ ID NOs: 2-972 and poly(2-oxazoline). In some embodiments of the methods disclosed herein, the present disclosure provides a therapeutic biomaterial comprising a recombinant collagen fragment having at least about 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, wherein the recombinant collagen fragment has the amino acid sequence set forth in any one of SEQ ID NOs: 974-1002, and poly(2-oxazoline).

[0133] In some embodiments of the methods disclosed herein, the present disclosure provides a therapeutic biomaterial comprising a recombinant collagen fragment having at least about 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 and polyethyleneimine. In some embodiments of the methods disclosed herein, the present disclosure provides a therapeutic biomaterial comprising a recombinant collagen fragment having a sequence comprising the amino acid sequence set forth in any one of SEQ ID NOs: 2-972 and polyethyleneimine. In some embodiments of the methods disclosed herein, the present disclosure provides a therapeutic biomaterial comprising a recombinant collagen fragment having at least about 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, wherein the recombinant collagen fragment has the amino acid sequence set forth in any one of SEQ ID NOs: 974-1002, and polyethyleneimine.

[0134] In some embodiments of the methods disclosed herein, the present disclosure provides a therapeutic biomaterial comprising a recombinant collagen fragment having at least about 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 and carboxymethylcellulose. In some embodiments of the methods disclosed herein, the present disclosure provides a therapeutic biomaterial comprising a recombinant collagen fragment having a sequence comprising the amino acid sequence set forth in any one of SEQ ID NOs: 2 to 972 and carboxymethylcellulose. In some embodiments of the methods disclosed herein, the present disclosure provides a therapeutic biomaterial comprising a recombinant collagen fragment having at least about 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, wherein the recombinant collagen fragment has the amino acid sequence set forth in any one of SEQ ID NOs: 974 to 1002, and carboxymethylcellulose.

[0135] In some embodiments of the methods disclosed herein, the present disclosure provides a therapeutic biomaterial comprising a recombinant collagen fragment having at least about 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 and chondroitin sulfate. In some embodiments of the methods disclosed herein, the present disclosure provides a therapeutic biomaterial comprising a recombinant collagen fragment having a sequence comprising the amino acid sequence set forth in any one of SEQ ID NOs: 2 to 972 and chondroitin sulfate. In some embodiments of the methods disclosed herein, the present disclosure provides a therapeutic biomaterial comprising a recombinant collagen fragment having at least about 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, wherein the recombinant collagen fragment has the amino acid sequence set forth in any one of SEQ ID NOs: 974 to 1002, and chondroitin sulfate.

[0136] In some embodiments of the methods disclosed herein, the present disclosure provides a therapeutic biomaterial comprising a recombinant collagen fragment having at least about 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 and acetylated hyaluronic acid. In some embodiments of the methods disclosed herein, the present disclosure provides a therapeutic biomaterial comprising a recombinant collagen fragment having a sequence comprising the amino acid sequence set forth in any one of SEQ ID NOs: 2 to 972 and acetylated hyaluronic acid. In some embodiments of the methods disclosed herein, the present disclosure provides a therapeutic biomaterial comprising a recombinant collagen fragment having at least about 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, wherein the recombinant collagen fragment has the amino acid sequence set forth in any one of SEQ ID NOs: 974 to 1002 and acetylated hyaluronic acid.

[0137] In some embodiments of the methods disclosed herein, the present disclosure provides a therapeutic biomaterial comprising a recombinant collagen fragment having at least about 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 and zinc hyaluronate. In some embodiments of the methods disclosed herein, the present disclosure provides a therapeutic biomaterial comprising a recombinant collagen fragment having a sequence comprising the amino acid sequence set forth in any one of SEQ ID NOs: 2 to 972 and zinc hyaluronate. In some embodiments of the methods disclosed herein, the present disclosure provides a therapeutic biomaterial comprising a recombinant collagen fragment having at least about 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, wherein the recombinant collagen fragment has the amino acid sequence set forth in any one of SEQ ID NOs: 974 to 1002 and zinc hyaluronate.

[0138] In some embodiments, the polydispersity of the collagen fragments disclosed herein can be about 1. In some embodiments, the polydispersity of the collagen fragments disclosed herein can range from about 1 to about 2. In some embodiments, the polydispersity of the collagen fragments disclosed herein can range from about 1 to about 3 or from about 1 to about 5.

[0139] In some embodiments, the pharmaceutically acceptable scaffold can be for use in the treatment of a subject in need of cancer treatment. In some embodiments, the cancer is characterized by the presence of a solid tumor. In some embodiments, the cancer can be skin, colon, oral mucosa, rectum, esophagus, thyroid, liver, pancreas, kidney, bladder, lung, brain, breast, ovary, testis, prostate, bone cancer, or any combination of the foregoing. In some embodiments, the biomaterial can be directly adhered onto cancer cells.

[0140] In some embodiments of the methods described herein, the biomaterial can comprise a recombinant collagen fragment that may not be hydroxylated. In some embodiments of the methods described herein, the biomaterial can comprise a recombinant collagen fragment that may be hydroxylated.

[0141] In some embodiments, the biomaterial can be for use in the treatment of a wound resulting from the excision of a primary tumor. In some embodiments, the biomaterial can be directly adhered onto the wound. In some embodiments, the biomaterial can be for use in maintaining cell quiescence after the excision of a primary tumor. In some embodiments, the biomaterial can be for use in maintaining cell quiescence in a subject who has not undergone excision of a primary tumor.

[0142] In some embodiments, the biomaterial can be directly adhered onto cells. Without wishing to be bound by a particular theory, it is believed that by directly placing a therapeutic biomaterial comprising a recombinant collagen fragment onto a wound or cell surface, type III collagen is concentrated in the ECM, and metastasis is restricted or prevented through induction of disseminated tumor cell quiescence.

[0143] In some embodiments, the therapeutic biomaterial can be a protein-polyurethane alloy. In some embodiments, the protein can be any of the collagen fragments (or combinations thereof) described herein, and the fragments move from the alloy to contact the cell surface when the alloy is exposed to water, buffer solution, and / or the cell surface.

[0144] In some embodiments, the alloy can comprise from about 10 wt% to about 50 wt% collagen fragments and from about 50 wt% to about 90 wt% polyurethane. In some embodiments, the alloy can comprise from about 20 wt% to about 35 wt% protein and from about 65 wt% to about 80 wt% polyurethane. In some embodiments, the alloy may or may substantially not contain particles of recombinant collagen fragments having an average diameter greater than 1 micron. In some embodiments, the protein-polyurethane alloy can be mechanically adhered using, for example, sutures or adhesives, or can be directly adhered onto the cell surface in a subject having cancer. In some embodiments, the protein-polyurethane alloy can be applied as a dry film or powder and can be hydrated naturally by the subject, or can be hydrated in situ after application using a suitable water or buffer. In embodiments where the protein-polyurethane alloy, or the hyaluronic acid-protein alloy, is applied to the cell surface as a dry film or powder, without wishing to be bound by a particular theory, it is believed that after application, the film or powder is hydrated by the cell surface. Alternatively, the film or powder can be hydrated by the application of a suitable buffer or water. In some embodiments, the film can be prepared by drying any of the alloys described herein using any standard drying method known in the art, such as a continuous oven. The powder form can be prepared by spray drying or freeze drying a solution, by grinding a pre-dried alloy, or by any other preparation method known in the art. Again, without wishing to be bound by a particular theory, it is believed that by directly placing any of the alloys described herein containing recombinant collagen fragments onto the cell surface, type III collagen is concentrated in the ECM, thereby restricting or preventing metastasis through induction of disseminated tumor cell dormancy.

[0145] In some embodiments, the pharmaceutically acceptable scaffold may comprise a protein hyaluronic acid alloy. In some embodiments, the alloy may comprise from about 10 wt% to about 50 wt% of the fragment and from about 50 wt% to about 90 wt% of hyaluronic acid. In some embodiments, the alloy may comprise from about 20 wt% to about 35 wt% of the protein and from about 65 wt% to about 80 wt% of hyaluronic acid. In some embodiments, hyaluronic acid or the protein, or both, and the protein are cross-linked either to themselves, to each other, or to both. In some embodiments, the protein may be dissolved, suspended, or present in the cross-linked hyaluronic acid matrix. In some embodiments, when the alloy is exposed to water or a buffer solution, the protein migrates from the alloy and contacts the cell surface.

[0146] In some embodiments, the pharmaceutically acceptable scaffold may comprise a protein dissolved in a gelatin matrix. In some embodiments, the alloy may comprise from about 10 wt% to about 50 wt% of the fragment and from about 50 wt% to about 90 wt% of gelatin. In some embodiments, the alloy may comprise from about 20 wt% to about 35 wt% of the protein and from about 65 wt% to about 80 wt% of gelatin. In some embodiments, the gelatin may be cross-linked. In some embodiments, when the alloy is exposed to water or a buffer solution, the protein migrates from the alloy and contacts the cell surface.

[0147] In some embodiments, the present disclosure provides a method of doing so in a subject in need of treating cancer, the method comprising contacting the cell surface of the subject with a therapeutic biomaterial comprising a pharmaceutically acceptable scaffold and an effective amount of recombinant collagen having the amino acid sequence set forth in SEQ ID NO: 973.

[0148] The amino acid sequence of SEQ ID NO: 973 is as follows:

[0149] In some embodiments, the present disclosure provides a method of inhibiting the growth of cancer cells, the method comprising contacting the cancer cells with a therapeutic biomaterial comprising a pharmaceutically acceptable scaffold and a recombinant collagen fragment having the amino acid sequence set forth in SEQ ID NO: 973.

[0150] In some embodiments, the present disclosure provides a method of doing so in a subject in need of inducing cell quiescence, the method comprising contacting the cell surface in the subject with a therapeutic biomaterial comprising a pharmaceutically acceptable scaffold and recombinant collagen having the amino acid sequence set forth in SEQ ID NO: 973.

[0151] In some embodiments, the cancer may be characterized by the presence of a solid tumor. In some embodiments, the cancer can be cancer of the skin, colon, oral mucosa, rectum, esophagus, thyroid, liver, pancreas, kidney, bladder, lung, brain, breast, ovary, testis, prostate, bone, or any combination of the foregoing. In some embodiments, the recombinant collagen may not be hydroxylated. In some embodiments, the recombinant collagen may be hydroxylated.

[0152] In some embodiments of methods of using the recombinant collagen having the amino acid sequence set forth in SEQ ID NO: 973 provided herein, the biomaterial can be a protein-polyurethane alloy. In some embodiments of methods of using the recombinant collagen having the amino acid sequence set forth in SEQ ID NO: 973 provided herein, the biomaterial can comprise a pharmaceutically acceptable scaffold. In some embodiments, the biomaterial can be directly adhered onto the cancer cells.

[0153] The protein polyurethane alloy described herein contains a protein, such as any of the fragments disclosed herein including SEQ ID NOs: 1 to 1002, dissolved in polyurethane or a plurality of polyurethanes. In some embodiments, the protein polyurethane alloy described herein contains a collagen fragment having at least about 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 and / or 973. In some embodiments, the protein polyurethane alloy described herein has at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity or similarity to SEQ ID NO: 1 and / or 973 and contains a recombinant collagen fragment. In some embodiments, the protein polyurethane alloy described herein contains a collagen fragment having the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the protein polyurethane alloy described herein contains a collagen fragment having the amino acid sequence set forth in any one of SEQ ID NOs: 2 to 972. In some embodiments, the protein polyurethane alloy described herein contains recombinant collagen having the amino acid sequence set forth in SEQ ID NO: 973, dissolved in polyurethane or a plurality of polyurethanes. In some embodiments, the protein polyurethane alloy described herein may contain recombinant collagen having the amino acid sequence set forth in any one of SEQ ID NOs: 974 to 1002, dissolved in polyurethane or a plurality of polyurethanes.

[0154] In certain embodiments, the protein polyurethane alloys described herein may include only one of the plurality of phases of polyurethane or a protein that is miscible with a plurality of polyurethanes in which the protein is blended. For example, in some embodiments, the protein polyurethane alloy may include only the hard phase of the polyurethane or a protein that is miscible with a plurality of polyurethanes having both a hard phase and a soft phase. The protein polyurethane alloys described herein may not include, or may substantially not include, proteins in the form of particles dispersed in the polyurethane. For example, in some embodiments, the protein polyurethane alloy may not include, or may substantially not include, protein particles having an average diameter greater than 1 micron (μm). In some embodiments, the protein polyurethane alloy may not include, or may substantially not include, particles of recombinant collagen fragments having an average diameter greater than 1 micron (μm). In some embodiments, the polyurethane may also include urea linkages. In such embodiments, the polyurethane is a polyurethane urea.

[0155] In some embodiments, the protein for use in a therapeutic biomaterial comprising a protein polyurethane alloy can be a succinylated protein. A succinylated protein is a protein modified by the addition of a succinyl group to the side chain of an amino acid in the protein. Most commonly, the succinyl group is added to the lysine side chain. The method of adding a succinyl group to the side chain of an amino acid in a protein is generally referred to as protein succinylation. The addition of a succinyl group to a protein can change the functional and structural properties of the protein. In some cases, the addition of a relatively large modification such as a succinyl moiety can be expected to change the tertiary structure of the protein. Further, the addition of the succinyl moiety can change the lysine side chain from a primary amine to an acid, making it more hydrophilic and changing its charge from positive to negative at physiological pH. Succinylation can be achieved using techniques and chemistries well known in the art.

[0156] Suitable polyurethanes for use in therapeutic biomaterials comprising the protein polyurethane alloy described in this specification include those comprising at least two phases including a "soft phase" and a "hard phase". The soft phase is formed from polyol segments within the polyurethane that are separate from the urethane-containing phase due to differences in polarity. The urethane-containing phase is referred to as the hard phase. This phase separation is well known in the art and is the basis for many properties of polyurethanes.

[0157] The soft phase is typically an elastomer at room temperature and typically has a softening point or glass transition temperature (Tg) below room temperature. Tg can be measured by Dynamic Mechanical Analysis (DMA) and quantified by either the peak of tan(δ) or the onset of the drop in storage modulus. Alternatively, Tg can be measured by Differential Scanning Calorimetry (DSC). In some cases, there may be crystallinity in the soft phase, which can typically be seen as a melting point between 0 °C and about 60 °C.

[0158] The hard phase typically has a Tg or melting point higher than room temperature, more typically higher than about 80 °C. The softening of the hard phase can be measured by measuring the onset of the drop in storage modulus (also referred to as stiffness) measured by DMA.

[0159] The "soft phase" of a polyurethane or a protein-polyurethane alloy containing polyurethane contains the polyol component of the polyurethane. Its function is to be soft and flexible at a temperature above its Tg in order to impart toughness, extensibility, and flexibility to the polyurethane. Typical soft segments can include polyether polyols, polyester polyols, polycarbonate polyols, and mixtures thereof. The soft segments typically range in molecular weight from about 250 D to greater than about 5 kD. The "hard phase" of a polyurethane or a protein-polyurethane alloy containing polyurethane contains the urethane segments of the polymer imparted by the isocyanate used to connect the polyol with short-chain diols such as butanediol and propanediol. In some embodiments, the chain extender can include amines such as ethylenediamine and hexamethylenediamine to introduce urea bonds. Urea bonds can also be introduced by adding water that reacts with the isocyanate to produce an amine and carbon dioxide that can act as a blowing agent. Thus, the term polyurethane is understood to include urea bonds in the hard phase in some embodiments. Typical isocyanates useful for this polyurethane include, but are not limited to, hexamethylene diisocyanate, isophorone diisocyanate, methylene diisocyanate, toluene diisocyanate, phenyl diisocyanate, etc. These molecules are more polar and rigid than the polyols used to make the soft segments. Thus, the hard segments are more rigid and have a higher softening point compared to the soft segments. The function of the hard phase is to provide strength, temperature resistance, and abrasion resistance to the polyurethane, among other properties.

[0160] In some embodiments described herein, the protein may be miscible only with the hard phase and substantially not change the soft phase transition. Without wishing to be bound by a particular theory, it is believed that when the protein is dissolved in the hard phase, it significantly increases the temperature at which the hard phase begins to soften, and thus increases the temperature resistance of the alloy.

[0161] In a protein-polyurethane alloy comprising one or more miscible proteins and a polyurethane, one or more proteins may be dissolved within the hard phase of the one or more polyurethanes. The protein-polyurethane alloy may include at least one protein that is miscible with the hard phase of the one or more polyurethanes in the alloy. In some embodiments, the protein-polyurethane alloy may include a plurality of proteins and / or a plurality of polyurethane hard phases that are miscible with each other. In all of these embodiments, without wishing to be bound by a particular theory, the protein or proteins are thought to be dissolved within the polyurethane or polyurethanes' hard phase.

[0162] One or more proteins dissolved within the hard phase of the one or more polyurethanes can form a homogeneous mixture when blended. In some embodiments, the protein-polyurethane alloy may include a plurality of proteins dissolved within the one or more polyurethanes such that the proteins and the polyurethanes form a homogeneous mixture when blended and dried. Typically, a protein-polyurethane alloy comprising a homogeneous mixture of protein and polyurethane does not include a significant amount of protein that is not dissolved in the polyurethane. That is, in some embodiments, the protein-polyurethane alloy may include fragments of protein dispersed within the polyurethane.

[0163] Suitable polyurethanes according to the embodiments described herein include, but are not limited to, aliphatic polyurethanes, aromatic polyurethanes, bio-based polyurethanes, or acrylic acid-modified polyurethanes. Suitable polyurethanes are commercially available from manufacturers including Covestro, Lubrizol, Hauthaway, Stahl, etc. In some embodiments, the polyurethane of the protein polyurethane alloy can be a biopolyurethane. In some embodiments, the polyurethane is a water-dispersible polyurethane. In some embodiments, the polyurethane can be a polyester polyurethane. In some embodiments, the polyurethane can be a polyether polyurethane. In some embodiments, the polyurethane can be a polycarbonate-based polyurethane. In some embodiments, the polyurethane can be an aliphatic polyester polyurethane. In some embodiments, the polyurethane can be an aliphatic polyether polyurethane. In some embodiments, the polyurethane can be an aliphatic polycarbonate polyurethane. In some embodiments, the polyurethane can be an aromatic polyester polyurethane. In some embodiments, the polyurethane can be an aromatic polyether polyurethane. In some embodiments, the polyurethane can be an aromatic polycarbonate polyurethane.

[0164] In some embodiments, the polyurethane can have a soft segment selected from the group consisting of polyether polyols, polyester polyols, polycarbonate polyols, and mixtures thereof. In some embodiments, the polyurethane can have a hard segment that includes a diisocyanate and optionally a short-chain diol or diamine. Suitable diisocyanates can be selected from the group consisting of aliphatic diisocyanates such as hexamethylene diisocyanate, isophorone diisocyanate; aromatic diisocyanates such as 4,4'-diphenylmethane diisocyanate, toluene diisocyanate, phenyl diisocyanate, and mixtures thereof. Suitable short-chain diols include ethylene glycol, propanediol, butanediol, 2,2-methyl-1,3-propanediol, pentanediol, hexanediol, and mixtures thereof. Suitable short-chain diamines include ethylenediamine, hexamethylenediamine, and mixtures thereof. In some embodiments, crosslinking agents such as polyfunctional alcohols (e.g., trimethylolpropane triol), or diamines such as ethylenediamine or 4,4'-diamino, diphenyldiamine.

[0165] Exemplary commercially available polyurethanes include, but are not limited to, L3360 and Hauthane HD-2001 available from C.L. Hauthaway & Sons Corporation, SANCURE™ polyurethanes, BONDTHANE™ polyurethanes available from Lubrizol Corporation, such as UD-108, UD-250, and UD-303 available from Bond Polymers International, EPOTAL® ECO 3702 and EPOTAL® P100 ECO from BASF, and Permutex Evo EX-RC-2214 (RC-2214) from Stahl. L3360 is an aliphatic polyester polyurethane polymer aqueous dispersion having 35% solids, a viscosity of 50 - 500 cps (centipoise), and a density of approximately 8.5 lb / gal (pounds per gallon). HD-2001 is an aliphatic polyester polyurethane polymer aqueous dispersion having 40% solids, a viscosity of 50 - 500 cps, and a density of approximately 8.9 lb / gal. BONDTHANE™ UD-108 is an aliphatic polyether polyurethane polymer aqueous dispersion having 33% solids, a viscosity of 300 cps, and a density of 8.7 lb / gal. BONDTHANE™ UD-250 is an aliphatic polyester polyurethane polymer aqueous dispersion having 35% solids, a viscosity of 200 cps, and a density of 8.8 lb / gal. BONDTHANE™ UD-303 is an aliphatic polyether polyurethane polymer aqueous dispersion having 35% solids, a viscosity of less than 500 cps, and a density of 8.7 lb / gal. EPTOAL® P100 ECO is a polyester polyurethane elastomer aqueous dispersion having approximately 40% solids and a viscosity of approximately 40 mPas. RC-2214 is an aliphatic polyether polyurethane polymer aqueous dispersion having 58 - 60% solids, a viscosity of 4,000 - 15,000 cps, and a density of approximately 8.9 lb / gal.

[0166] Exemplary bio-based polyurethanes include, but are not limited to, L3360 available from C.L. Hauthaway & Sons Corporation, IMPRANIL® Eco DLS, IMPRANIL® Eco DL519, IMPRANIL® Eco DLP-R, and IMPRAPERM® DL 5249 available from Covestro. IMPRANIL® Eco DLS is an anionic aliphatic polyester polyurethane polymer aqueous dispersion having approximately 50% solids content, a viscosity of less than 1,200 MPa·s, and a density of about 1.1 g / cc. IMPRANIL® Eco DL 519 is an anionic aliphatic polyester polyurethane polymer aqueous dispersion. IMPRANIL® Eco DLP-R is an anionic aliphatic polyester polyurethane polymer aqueous dispersion. IMPRAPERM® DL 5249 is an anionic aliphatic polyester-polyurethane polymer aqueous dispersion.

[0167] In some embodiments, the polyurethane may include reactive groups that can be crosslinked with a protein. Exemplary reactive groups include, but are not limited to, sulfonates, aldehydes, carboxylic acids or esters, or blocked isocyanates, and combinations thereof. In such embodiments, the polyurethane can be crosslinked to the protein in the protein-polyurethane alloy through the reaction of reactive groups on the protein with reactive groups present in the polyurethane.

[0168] In some embodiments, the amount of protein (a recombinant collagen fragment having at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity or similarity to SEQ ID NO: 1 and / or 973, or a collagen fragment having the amino acid sequence set forth in any one of SEQ ID NOs: 2-972, or a recombinant collagen having the amino acid sequence set forth in SEQ ID NO: 973, or a recombinant collagen having the amino acid sequence set forth in any one of SEQ ID NOs: 974-1002) in the protein polyurethane alloy can range from about 5 wt% to about 50 wt% of the protein, including sub-ranges. For example, in some embodiments, the amount of protein in the polyurethane alloy can be in the range of about 5 wt% to about 50 wt%, about 10 wt% to about 50 wt%, about 15 wt% to about 50 wt%, about 20 wt% to about 50 wt%, about 25 wt% to about 50 wt%, about 30 wt% to about 50 wt%, about 35 wt% to about 50 wt%, about 40 wt% to about 50 wt%, about 45 wt% to about 50 wt%, about 5 wt% to about 45 wt%, about 5 wt% to about 40 wt%, about 5 wt% to about 35 wt%, about 5 wt% to about 30 wt%, about 5 wt% to about 25 wt%, about 5 wt% to about 20, about 5 wt% to about 15 wt%, or about 5 wt% to about 10 wt%, or a range within a range having any two of these values including the endpoints as endpoints. In some embodiments, the amount of protein in the protein polyurethane alloy can range from about 20 wt% to about 35 wt%.

[0169] In some embodiments, the amount of polyurethane in the protein-polyurethane alloy can range from about 50 wt% to about 95 wt%, including sub-ranges. For example, in some embodiments, the amount of polyurethane in the protein-polyurethane alloy is from about 50 wt% to about 95 wt%, from about 55 wt% to about 95 wt%, from about 60 wt% to about 95 wt%, from about 65 wt% to about 95 wt%, from about 70 wt% to about 95 wt%, from about 75 wt% to about 95 wt%, from about 80 wt% to about 95 wt%, from about 85 wt% to about 95 wt%, from about 90 wt% to about 95 wt%, from about 50 wt% to about 90 wt%, from about 50 wt% to about 85 wt%, from about 50 wt% to about 80 wt%, from about 50 wt% to about 75 wt%, from about 50 wt% to about 70 wt%, from about 50 wt% to about 65 wt%, from about 50 wt% to about 60 wt%, or from about 50 wt% to about 55 wt%, or a range within a range having any two of these values as endpoints, including the endpoints. In some embodiments, the amount of polyurethane in the protein-polyurethane alloy can range from about 65 wt% to about 80 wt%.

[0170] Any of the ranges listed above may be combined for the weight percentages of the protein and polyurethane in the protein-polyurethane alloy. For example, in some embodiments, the weight percentages of the protein and polyurethane in the protein-polyurethane alloy may be any of the following. The amount of protein in the polyurethane alloy may range from about 5 wt% to about 50 wt%, and the amount of polyurethane in the protein-polyurethane alloy may range from about 50 wt% to about 95 wt%. The amount of protein in the polyurethane alloy may range from about 15 wt% to about 50 wt%, and the amount of polyurethane in the protein-polyurethane alloy may range from about 50 wt% to about 85 wt%. The amount of protein in the polyurethane alloy may range from about 20 wt% to about 50 wt%, and the amount of polyurethane in the protein-polyurethane alloy may range from about 50 wt% to about 80 wt%. The amount of protein in the polyurethane alloy may range from about 25 wt% to about 50 wt%, and the amount of polyurethane in the protein-polyurethane alloy may range from about 50 wt% to about 75 wt%. The amount of protein in the polyurethane alloy may range from about 30 wt% to about 50 wt%, and the amount of polyurethane in the protein-polyurethane alloy may range from about 50 wt% to about 70 wt%. The amount of protein in the polyurethane alloy may range from about 10 wt% to about 40 wt%, and the amount of polyurethane in the protein-polyurethane alloy may range from about 60 wt% to about 90 wt%. The amount of protein in the polyurethane alloy may range from about 15 wt% to about 40 wt%, and the amount of polyurethane in the protein-polyurethane alloy may range from about 60 wt% to about 85 wt%. The amount of protein in the polyurethane alloy may range from about 20 wt% to about 40 wt%, and the amount of polyurethane in the protein-polyurethane alloy may range from about 60 wt% to about 80 wt%. The amount of protein in the polyurethane alloy may range from about 20 wt% to about 35 wt%, and the amount of polyurethane in the protein-polyurethane alloy may range from about 65 wt% to about 80 wt%. In some embodiments, the weight percentage values and ranges listed above may be based on the total weight of the protein-polyurethane alloy.In some embodiments, the weight percent values and ranges listed above may be based on the total weight of only the protein and polyurethane in the protein-polyurethane alloy. Unless otherwise specified, the weight percent values or ranges of polyurethane and protein are based on the total weight of only the protein and polyurethane in the protein-polyurethane alloy.

[0171] In some embodiments, the total amount of protein (a recombinant collagen fragment having at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity or similarity to SEQ ID NO: 1 and / or 973, or a collagen fragment having the amino acid sequence set forth in any one of SEQ ID NOs: 2-972, or a recombinant collagen having the amino acid sequence set forth in SEQ ID NO: 973) and polyurethane in the protein-polyurethane alloy can be about 80 wt% or more. For example, in some embodiments, the total amount of protein and polyurethane in the protein-polyurethane alloy is in the range of about 80 wt% to 100 wt%, about 82 wt% to 100 wt%, about 84 wt% to 100 wt%, about 86 wt% to 100 wt%, about 88 wt% to 100 wt%, about 90 wt% to 100 wt%, about 92 wt% to 100 wt%, about 94 wt% to 100 wt%, about 96 wt% to 100 wt%, or about 98 wt% to 100 wt%.

[0172] In some embodiments, the protein polyurethane alloy may contain water that constitutes a portion of the total weight percentage of the material. In some embodiments, the amount of water in the protein polyurethane alloy can range from about 1 wt% to about 10 wt%, including sub - ranges. For example, in some embodiments, the amount of water in the protein polyurethane alloy is from about 1 wt% to about 10 wt%, from about 2 wt% to about 10 wt%, from about 3 wt% to about 10 wt%, from about 4 wt% to about 10 wt%, from about 5 wt% to about 10 wt%, from about 6 wt% to about 10 wt%, from about 7 wt% to about 10 wt%, from about 8 wt% to about 10 wt%, from about 1 wt% to about 9 wt%, from about 1 wt% to about 8 wt%, from about 1 wt% to about 7 wt%, from about 1 wt% to about 6 wt%, from about 1 wt% to about 5 wt%, from about 1 wt% to about 4 wt%, or from about 1 wt% to about 3 wt%, or a range within a range having any two of these values including the endpoints as endpoints.

Example

[0173] The examples and embodiments described herein are for illustrative purposes only, and it is understood that various modifications or changes suggested thereby to those skilled in the art are within the spirit and scope of this application.

[0174] Example 1: Effective Coagulation of Recombinant Collagen Protein in Polyurethane To determine whether recombinant collagen can be effectively coagulated with commercially available polyurethane dispersions designed for medical use, a 50 kDa recombinant collagen protein ( "50 kDa rCol") containing the amino acid sequence of SEQ ID NO: 1 was formulated in various polyurethane compositions as described below.

[0175] 50kDa rCol: Baymedix® AD111 The sample was prepared by dissolving 0.86 g of 50KDa rCol in 4.29 mL of deionized water and stirring with a magnetic stir bar at 600 rpm for 30 minutes at 20 °C. After stirring for 30 minutes, 6.86 g of Baymedix® AD111 (Covestro AG, Leverkusen, Germany) polyurethane dispersion was added to the solution and stirred at 600 rpm for 30 minutes. Next, the solution of polyurethane and 50KDa rCol was pipetted into a 10 cm diameter Teflon® evaporating dish. The dish was dried overnight in an oven at 45 °C. After drying, the dried solution was conditioned in a standard reference atmosphere (23 °C, 50% humidity) for 24 hours to produce a 50KDa rCol-AD111 coagulation film.

[0176] The films were tested using a DMA-850 from TA Instruments. Strips 1 cm × 2.5 cm were cut from each film using a metal die. The cut film samples were loaded into the film and fiber tension clamps for testing. During the test, a preload of 0.01 N was applied to the cut film samples. The instrument was cooled to -80 °C and held for 1 minute, then the temperature was raised at 4 °C / min to 200 °C or until the sample was too weak to hold tension. During the temperature ramp, the samples were vibrated at a frequency of 1 Hz with a strain of 0.1%. The storage modulus, loss modulus, and tan(δ) were plotted against temperature for both films. The resulting second storage modulus transition (obtained as the starting point of the last decrease in the measured storage modulus, i.e., the second DMA modulus transition onset temperature) was 125.0 °C for the 50KDa rCol-AD111 film. The control sample of AD111 was too soft for the DMA test.

[0177] In addition, four tensile specimens (by Standard Test Method for Tensile Properties of Plastics “ASTM D638”) were each cut from the sample film dried and conditioned using a metal die. The cut film specimens were loaded into an INSTRON® 5960 (Instron, Norwood, MA) series machine and pulled with a tension of 100 millimeters per minute until rupture. The average Young's modulus, average tensile strength (maximum tensile stress), and average elongation at break were recorded. For the 50KDa rCol-AD111 film, the Young's modulus was 131 MPa, the maximum tensile stress was 4.8 MPa, and the elongation at break was 209%.

[0178] 50kDa rCol:Baymedix CD102 The sample was prepared by dissolving 0.75 g of 50KDa rCol in 3.75 mL of deionized water and stirring with a magnetic stir bar at 600 rpm for 30 minutes at 20 °C. After stirring for 30 minutes, 7.5 g of Baymedix® CD102 (Covestro AG, Leverkusen, Germany) polyurethane dispersion was added to the solution and stirred at 600 rpm for 30 minutes. Then, the solution of polyurethane and 50KDa rCol was pipetted into a 10 cm diameter Teflon® evaporating dish. The dish was dried overnight in an oven at 45 °C. After drying, the dried solution was conditioned in a standard reference atmosphere (23 °C, 50% humidity) for 24 hours to produce a 50KDa rCol polyurethane coagulation film.

[0179] The dynamic mechanical analysis (DMA) test was performed as outlined above. The second storage modulus transition that occurred in the 50KDa rCol-CD102 film (obtained as the starting point of the last decrease in the measured storage modulus, i.e., the second DMA modulus transition start temperature) was 155.9 °C, an increase of 27.3 °C (128.6 °C) compared to the CD102 control sample.

[0180] The tensile test was carried out as outlined above. For the 50KDa rCol-CD102 film, the average Young's modulus was 48 MPa, the measured average tensile stress was 13 MPa, and the average elongation at break was 754%.

[0181] 50kDa rCol:Baymedix® CD104 The sample was prepared by dissolving 1.25 g of 50KDa rCol in 6.25 mL of deionized water and stirring with a magnetic stir bar at 600 rpm for 30 minutes at 20 °C. After stirring for 30 minutes, 12.5 g of Baymedix® CD104 (Covestro AG, Leverkusen, Germany) polyurethane dispersion was added to the solution and stirred at 600 rpm for 30 minutes. Then, the solution of polyurethane and 50KDa rCol was pipetted into a 10 cm diameter Teflon® evaporating dish. The dish was dried overnight in an oven at 45 °C. After drying, the dried solution was conditioned in a standard reference atmosphere (23 °C, 50% humidity) for 24 hours to produce a 50KDa rCol polyurethane coagulation film.

[0182] The DMA test was carried out as outlined above. The second storage modulus transition that occurred in the 50KDa rCol-CD104 film (obtained as the starting point of the last decrease in the measured storage modulus, i.e., the second DMA modulus transition onset temperature) was 109.7 °C, an increase of 37.8 °C (71.9 °C) compared to the CD104 control sample.

[0183] The tensile test was carried out as outlined above. For the 50KDa rCol-CD104 film, the average Young's modulus was 150 MPa, the measured average tensile stress was 19 MPa, and the average elongation at break was 598%.

[0184] 50kDa rCol:Baymedix® FD103 The sample was prepared by dissolving 0.94 g of 50KDa rCol in 4.72 mL of deionized water and stirring with a magnetic stir bar at 600 rpm for 30 minutes at 20°C. After stirring for 30 minutes, 6.34 g of Baymedix® FD103 (Covestro AG, Leverkusen, Germany) polyurethane dispersion was added to the solution and stirred at 600 rpm for 30 minutes. Subsequently, the solution of polyurethane and 50KDa rCol was pipetted into a 10 cm diameter Teflon® evaporating dish. The dish was dried overnight in an oven at 45°C. After drying, the dried solution was conditioned in a standard reference atmosphere (23°C, 50% humidity) for 24 hours to produce a 50KDa rCol polyurethane coagulation film.

[0185] The DMA test was conducted as outlined above. The second storage modulus transition (obtained as the starting point of the last decrease in the measured storage modulus, i.e., the second DMA modulus transition onset temperature) that occurred in the 50KDa rCol-FD103 film was 178.1°C, an increase of 14.2°C compared to the FD103 control sample (163.9°C).

[0186] The tensile test was conducted as outlined above. In the 50KDa rCol-FD103 film, the average Young's modulus was 45 MPa, the measured average tensile stress was 19 MPa, and the average elongation at break was 414%.

[0187] Baymedix® FD103 foam The sample is prepared by mechanical foaming of Baymedix® FD103. After foaming, depending on the thickness of the foam, the foam is dried at a high temperature (e.g., 120°C) for a sufficient length of time (e.g., 10 - 30 minutes). Subsequently, the dried foam is immersed in a 50kDa rCol deionized aqueous solution at a specific concentration (e.g., 200 mg / ml).

[0188] Baymedix® FD103:AD111 foam The sample is prepared by mechanically foaming a mixture of Baymedix® FD103:AD111. After foaming, depending on the thickness of the foam, the foam is dried at a high temperature (e.g., 120 °C) for a sufficiently long time (e.g., 10 - 30 minutes). Then, the dried foam is immersed in a 50 kDa rCol deionized aqueous solution at a certain concentration (e.g., 200 mg / ml).

[0189] 50 kDa rCol: Baymedix® FD103 foam The sample is prepared by mechanically foaming a mixture of a 50 kDa rCol deionized aqueous solution and Baymedix® FD103. After foaming, depending on the thickness of the foam, the foam is dried at a high temperature (e.g., 120 °C) for a sufficiently long time (e.g., 10 - 30 minutes). Then, the dried foam is immersed in a 50 kDa rCol deionized aqueous solution at a certain concentration (e.g., 200 mg / ml).

[0190] 50 kDa rCol: Baymedix® FD103:AD111 foam A 16.7% (w / w) stock solution of 50 kDa rCol in water was prepared. Baymedix FD103 and Baymedix® AD111 are each aqueous polyurethane dispersions. A stock solution of Baymedix FD103:AD111 (Baymedix® FD103 and Baymedix® AD111) was prepared at a ratio of 70:30 based on the weight of the polyurethane dispersion. A 10% (w / w) stock solution of Rheolate208 (Elementis, London, UK) was prepared in water. 9.16 g of the recombinant collagen stock solution and 11.50 g of the Baymedix FD103:AD111 stock solution were mixed in a 50 mL beaker using an overhead mixer. Then, 1.55 g of the Rheolate208 stock solution was added and the solution was mixed further. Next, 0.72 g of ChemTex2216 (Cumberland, RI), 0.65 g of ChemTex2317, and 0.03 g of ChemTex2243 were added and the resulting mixture was mixed further. The resulting mixture was mechanically foamed by vigorously mixing using an overhead mixer. After foaming to introduce air bubbles, the foamed mixture was coated on a glass plate on a TQC film (TQC Automatic Film Applicator, Gardco) with a gap setting of 600 μm. The coating was dried at 75 °C for 30 minutes to obtain a porous foam sheet after drying.

[0191] This example demonstrates that a 50 kDa recombinant collagen protein containing the amino acid sequence of SEQ ID NO: 1 can effectively coagulate with a polyurethane dispersion designed for medical use.

[0192] 50kDa rCol:Hauthaway L3360 polyurethane dispersion The sample was prepared by dissolving 1 g of 50KDa rCol in 5 mL of deionized water and stirring with a magnetic stir bar at 1000 rpm for 1 hour at 20 °C. After stirring for 1 hour, 6.7 g of L3360 (Hauthaway L3360 Polyurethane Dispersion) was added to the solution and stirred at 1000 rpm for 30 minutes. Subsequently, the solution of polyurethane and 50KDa rCol was pipetted into a 10 cm diameter Teflon evaporating dish. The dish was dried overnight in an oven at 45 °C. After drying, the dried sample was conditioned in a standard reference atmosphere (23 °C, 50% humidity) for 24 hours to produce a 50KDa rCol polyurethane alloy film.

[0193] The film was tested using a DMA-850 from TA Instruments. The second storage modulus transition (obtained as the starting point of the last decrease in the measured storage modulus, i.e., the second DMA modulus transition onset temperature) that occurred in the 50KDa rCol polyurethane alloy was 177.8 °C, an increase of 62.9 °C compared to the control sample. The storage modulus measured in the control sample was 114.9 °C.

[0194] The tensile test was performed on an Instron® 5960 series machine filled with a tension of 100 millimeters per minute until it broke. The average Young's modulus was 161 MPa, the measured average tensile stress was 17 MPa, and the average elongation at break was 173% for the 50KDa rCol polyurethane alloy.

[0195] Example 2: In vivo evaluation of collagen-supplemented extracellular matrix The anti-cancer activity of the extracellular matrix (ECM) was evaluated by treating with commercially available bovine type III collagen (“bovine type III collagen”) (Uniprot sequence Q08E14; SEQ ID NO: 1009) and a 50 kDa recombinant collagen protein (SEQ ID NO: 1). The treatment solution of bovine type III collagen or SEQ ID NO: 1 was mixed with 4T1-Luc-1A4 mouse breast cancer cells and then subcutaneously implanted into female Balb / c mice. Tumors were collected at the end of life, photographed, and then bisected. The details of the experiment are the results below.

[0196] 4T1-Luc2-1A4 cells Mouse breast cancer cells (4T1-Luc2-1A4) were grown in Park Memorial Institute (RPMI) 1640 medium (ThermoFisher Scientific), 1 mM sodium pyruvate, 10 mM HEPES buffer, 2.8 mL of 45% glucose (1.25 g), 10% NHI fetal bovine serum (FBS), 1% PSG. Cells were dissociated with 0.25% trypsin / 2.21 mM EDTA in Hanks’ Balanced Salt Solution (HBSS) and 5.00E+05 trypan-excluding cells were resuspended in 200 μL of serum-free RPMI 1640 medium. During the preparation of the inoculum, collagen was added to the cells. Each inoculum was supplemented with 1.2 mL of collagen. Test samples were prepared at different concentrations as shown below. Bovine type III collagen was obtained from Nippi-inc (Japan; catalog number PSC-3-100-20).

[0197] Eight groups of animals (one control group and seven experimental groups; N = 8) were included in the study and treated as shown in Table 2. The inoculation dose for each animal was 5.00E+05 cells.

[0198]

Table 2

[0199] Each mouse was subcutaneously injected with 200 μL of the inoculum (right axilla (high)). All procedures performed in this experiment were conducted in accordance with the applicable laws, regulations, and guidelines of the National Institutes of Health (NIH). Cell viability was measured before and after injection as shown in Table 3.

[0200]

Table 3

[0201] On day 23, the tissues were excised, photographed, and the tumors were bisected. One half of the tumor was placed in 10% Neutral Buffered Formalin (NBF) (Sigma) for 24 hours and transferred to 70% EtOH for processing into FFPE blocks. Day 23 was selected for evaluation because it was the last day that all animals remained in the study. The blocks were stored at ambient temperature. The other half of the tumor was snap-frozen in liquid nitrogen. The tissue was stored at -80 °C. A summary of the group resistance to treatment and response to treatment is provided in Table 4.

[0202]

Table 4

[0203] Results All treatments were well tolerated and did not result in weight loss over the course of the study (Table 3). One mouse in Group 3 was found dead on day 28, and one mouse in Group 6 was euthanized on day 28 due to severe motor impairment. No other treated animals were euthanized for any reason other than reaching the maximum tumor volume imposed by the institutional animal care and use committee (IACUC) or the end of the study. The deaths seen in Groups 3 and 6 are not included in the conventional treatment window 14 days after treatment, but both animals are considered to have died within the treatment window due to the nature of the treatment implementation and its uncertain effects on disease progression.

[0204] Inoculation of 5.00E+05 cells per animal in the control group (Group 1) resulted in a median tumor doubling time of 5.0 days and a median tumor-free period of 25 days. All tumor growth evaluations were compared to Group 1 (untreated control) (Figures 1, 2A–2I, 3, and 4).

[0205] The addition of bovine collagen (Group 2) resulted in a median tumor doubling time of 5.8 days, a 60% median ΔT / ΔC value on day 23, and an increase in the time to progression (ITP) of 16.0%. There were no survivors without tumors in this group.

[0206] The addition of bovine collagen and 0.1% EtOH (Group 3), or bovine collagen and 0.01% EtOH (Group 4), resulted in median tumor doubling times of 6.2 and 6.6 days, median ΔT / ΔC values on day 23 of 70% and 58%, and ITP values of 8.0% and greater than 16.0%, respectively. There were no survivors without tumors in these groups.

[0207] Surprisingly, treatment with hydroxylated collagen did not show statistically significant tumor growth inhibition compared to the untreated control (data not shown). Without wishing to be bound by a particular theory, it is thought that residual ethanol in the hydroxylated collagen sample interfered with the experimental results. For example, reference is made to Alcohol Res. 2015;37(2):311-322 (2015), which is incorporated herein by reference and which explains that ethanol can induce tumor growth. To investigate the effect of ethanol, 0.1% and 0.01% ethanol were added to groups 3 and 4 (bovine collagen control) to determine the effect of ethanol on tumor size compared to groups 5-8. In all cases, the addition of ethanol induced tumor growth and suppressed the effect of bovine collagen as seen in group 2 (bovine collagen alone).

[0208] The addition of SEQ ID NO: 1 at 200 mg / mL, 10 mg / mL, or 1.5 mg / mL (groups 5, 6, and 7, respectively) resulted in a median tumor doubling time in the range of 5.5 - 6.2 days, a 23-day median ΔT / ΔC value in the range of 51% - 64%, and an ITP value in the range of 12.0% - 16.0% or greater. There were no survivors without tumors in these groups. SEQ ID NO: 1 was most potent at a dose of 200 mg / mL (group 5).

[0209] The addition of SEQ ID NO: 1 at 10 mg / mL (group 8) resulted in a median tumor doubling time of 5.1 days, a 23-day median ΔT / ΔC value of 75%, and an ITP value of 4.0%. There were no survivors without tumors in this group.

[0210] This example demonstrates that treatment with SEQ ID NO: 1 produces anti-cancer activity that is as effective as treatment with bovine collagen.

[0211] The effect of treatment on the estimated tumor volume is shown in FIGS. 2A - 2J. The tumor volume is described as "estimated" because it is an indirect measurement of the tumor volume. After each animal was euthanized and the tumor removed, the true tumor volume was calculated (FIGS. 1A, 1B, and 4).

[0212] Anatomical pathological analysis To understand the effect of SEQ ID NO: 1 on cancer dormancy, 4T1-Luc-1A4 mouse mammary tumor blocks stained with H&E and Masson's trichrome from female BALB / c mice in the above examples were analyzed for the relative amounts of viable tumor cells, necrosis, and collagen in the viable tumor regions compared to untreated samples. Tumor blocks were obtained from mice administered 5E+05 4T1-Luc1A4 cells / animal combined with control (Group 1), 1.5 mg / ml bovine type III collagen (Group 2), or 200, 10, or 1.5 mg / ml of SEQ ID NO: 1 (Groups 5, 6, and 7, respectively) via subcutaneous injection (200 μL) (the above experiment). Slides stained with H&E and Masson's trichrome were digitally scanned from three separate regions (beginning, middle, and end) of each tumor block, and the scanned images were examined and recorded (Table 4). Fourteen tumor blocks from the following animals were stained with H&E, Masson's trichrome, and immunohistochemistry (IHC).

[0213] All slides (H&E, Masson's trichrome, and immunohistochemistry (IHC)) were digitally scanned and evaluated at 40x magnification. Approximate percentage values for the relative areas of viable tumor cells, necrosis, and collagen within the viable tumor cell region were generated only via evaluation of the scanned images and are presented in Table 5.

[0214] [Table 5]

[0215] In all groups, regardless of the area of the tumor block, the area of necrosis was localized to the center of the tumor and, on average, constituted approximately 40 - 60% of the total tumor area in each group (Figures 5A - 5E). The area of viable tumor cells was mostly located along the periphery of the tumor and constituted approximately 40 - 60% of the total tumor area, regardless of the group or area of the tumor within the block. The groups administered 200 mg / ml of SEQ ID NO: 1, 10 mg / ml of SEQ ID NO: 1, and 1.5 mg / ml of SEQ ID NO: 1, or bovine collagen, had a higher overall average percentage of collagen in the viable tumor area compared to the control. Additionally, all three groups administered SEQ ID NO: 1 had a greater average percentage of collagen in the viable tumor area compared to the animals in Group 2 administered 1.5 mg / mL of bovine collagen. Representative micrographs of subcutaneous tumors from untreated and treated BALB / c mice are shown in Figures 6A - 6J.

[0216] Animals in Group 2 administered 1.5 mg / mL of bovine collagen had the lowest average percentage of collagen in the viable tumor area among the test article - treated groups (average 8.33 [±2.50], range 5 - 10). However, the average percentage of collagen in the viable tumor area was still higher than that of the untreated control animals.

[0217] Animals in Group 5 (200 mg / mL of SEQ ID NO: 1) had the highest relative average percentage of collagen in the viable tumor area compared to the control group and all other groups (average 10.56 [±1.67%], range 10 - 15%).

[0218] Animals in Group 6 (10 mg / mL of SEQ ID NO: 1) had a higher average relative percentage of collagen in the viable tumor percent area compared to the control animals, the animals in Group 2 administered 1.5 mg / mL of bovine collagen, and the animals in Group 7 administered 1.5 mg / ml of SEQ ID NO: 1 (average 10.00 [±2.50%], range 5 - 15).

[0219] Animals in Group 7 administered with 1.5 mg / mL of SEQ ID NO: 1 had a higher average percentage of collagen in the surviving tumors than the control animals and animals in Group 4 administered with 1.5 mg / mL of commercially available type III collagen (average 9.44 [±3.91%], range 5 - 15).

[0220] Mass spectrometry analysis of tumor tissue The extracted tumor tissue was evaluated by mass spectrometry, and the results are shown in Figures 7 and 8. This process is provided below.

[0221] Samples were loaded onto an LC-MS (Agilent 6545XT AdvanceBio LC / Q-TOF) for analysis. LC separation was achieved using an Agilent peptide mapping column (Agilent AdvanceBio Peptide Mapping 120Å, 2.1×150 mm, 2.7 μm, catalog number: 653750-902) flowing at a flow rate of 0.4 mL / min using a 140-minute gradient LC method. The gradient parameters were as follows (solvent A: 0.1% formic acid, solvent B: 80% acetonitrile, 0.1% formic acid): 0 - 3 minutes: 2 - 10% B, 3 - 107 minutes: 10 - 30% B, 107 - 121 minutes: 30 - 40% B, 121 - 126 minutes: 40 - 60% B, 126 - 127: 60 - 100% B, 127 - 137 minutes: 100% B, 137 - 138 minutes: 100 - 0% B, 138 - 140 minutes: 0% B.

[0222] Each sample was analyzed by LC-MS using a conventional automated MS / MS method (data-dependent mode). MS was performed at a resolution of approximately 30,000 over a range of 100 - 3000 m / z at an acquisition rate of 6 spectra / second. MS / MS analysis was performed on the top 8 precursor ions over a range of 50 - 3000 m / z at an acquisition rate of 3 spectra / second in each cycle using normalized collision energy (based on size and charge) and dynamic exclusion of 2 spectra released after 0.1 minute.

[0223] Peptide identification and quantification were achieved using Byologic software (Protein Metrics Inc., San Carlos, CA). All untrimmed Agilent * .d data files were searched against the Mus musculus (mouse) proteome protein database (Uniprot, Proteome ID: UP000000589) concatenated with SEQ ID NO: 1. Peptide search parameters were set for complete specific trypsin digestion, allowing a maximum of two cleavage misses. For the QTOF / HCD fragment type, the fragment mass error range was set to 20 ppm and the precursor mass error range was set to 15 ppm.

[0224] Carbamidomethylation (Cys; +57.021464 Da) was set as a fixed modification. The following were set as rare (one occurrence per peptide) variable modifications: Gln->pyro-Glu (N-terminal Gln; -17.026549 Da), Glu->pyro-Glu (N-terminal Glu; -18.010565), carbamylation (peptide N-terminus; +43.005814), phosphorylation (Ser, Thr, Tyr; +79.966331), and oxidation (Met; +15.994915). Deamidation (Asn; +0.984016) was set as a common variable modification. Hydroxyproline (up to three per peptide) and hydroxylysine were assigned as variable modifications only for "collagen family" proteins to improve the peptide application range.

[0225] After peptide identification and sequence matching, all scans with scores less than 100 were excluded from the dataset. The relative protein abundance for each sample was calculated by summing the XIC regions of all peptide matches for a specific protein and dividing by the total peptide XIC region for that sample. The relative protein abundances were then compared for the protein of interest between the experimental conditions and the negative (group 1) / positive (group 2) control groups.

[0226] As shown in Figure 8 and Table 5, collagen was enriched by more than 100% in each experimental group compared to the negative control.

[0227] The collagen composition of the tumor samples determined by mass spectrometry is described in Table 6.

[0228]

Table 6

[0229] This example demonstrates that treatment with bovine collagen and treatment with SEQ ID NO: 1 increase the amount of collagen in the tumor ECM. Treatment with SEQ ID NO: 1 is as effective as treatment with commercially available bovine type III collagen.

[0230] In Vitro Cancer Cell Line Survival Assay An in vitro cell survival assay was performed to evaluate whether SEQ ID NO: 1 (50 kDa rCol protein) induces dormancy in various cancers.

[0231] Fourteen different conditions (negative control (basal medium + 1.5% FBS), positive control (puromycin (500 μg / mL), six different SEQ ID NO: 1 concentrations, and six different bovine type III collagen concentrations) were compared. Cell lines 4T1, HCT116, Hep G2, and MCF7 were treated with SEQ ID NO: 1 and bovine collagen. The criteria for the selected cell lines included (1) a cell doubling time of 30 hours or less, (2) cells of human origin, and (3) cells that were able to grow on plastic without Matrigel ECM matrix. The levels of proliferation between different treatments were assayed as described and the results are shown in Figures 9A - 9F (bovine) and Figures 10A - 10C (hCOL3). Fourteen conditions were selected to evaluate each cell line. 1. Negative control - untreated (n = 6 minimum) 2. Positive control - puromycin (a potent translation inhibitor that causes rapid cell death) (n = 6 minimum) 3. 2% w / w of SEQ ID NO:1 (n = 6 minimum) [Final solution concentration] 4. 1% w / w of SEQ ID NO:1 (n = 6 minimum) [Final solution concentration] 5. 0.5% w / w of SEQ ID NO:1 (n = 6 minimum) [Final solution concentration] 6. 0.25% w / w of SEQ ID NO:1 (n = 6 minimum) [Final solution concentration] 7. 0.125% w / w of SEQ ID NO:1 (n = 6 minimum) [Final solution concentration] 8. 0.0625% w / w of SEQ ID NO:1 (n = 6 minimum) [Final solution concentration] 9. 2.7 mg / mL of bovine type III collagen (n = 6 minimum) [Final solution concentration] 10. 2 mg / mL of bovine type III collagen (n = 6 minimum) [Final solution concentration] 11. 1.5 mg / mL of bovine type III collagen (n = 6 minimum) [Final solution concentration] 12. 1 mg / mL of bovine type III collagen (n = 6 minimum) [Final solution concentration] 13. 0.5 mg / mL of bovine type III collagen (n = 6 minimum) [Final solution concentration] 14. 0.1 mg / mL of bovine type III collagen (n = 6 minimum) [Final solution concentration]

[0232] The results for bovine collagen are shown in FIGS. 9A-9F. Surprisingly, higher bovine collagen concentrations correlate with higher absorbance and suggest higher cell proliferation. Despite this data, lower bovine collagen concentrations (e.g., 1 mg / mL, 0.5 mg / mL, and 0.1 mg / mL) show reduced absorbance compared to higher concentrations and suggest reduced cell proliferation. The apparent discrepancy resulting from the bovine collagen concentration suggests that bovine collagen interferes with the assay and, despite the data presented in FIGS. 9A-9E at higher concentrations, rather, bovine collagen at 2.7 mg / mL, 2 mg / mL, and 1.5 mg / mL inhibits cell growth. This is supported by the data in FIG. 9F showing the effect of different concentrations of bovine collagen on 4T1 cells measured by the growth assay read at time zero. Furthermore, the microscopic images in FIG. 9E show that bovine collagen inhibits cell growth at all concentrations including the high concentration of 2.7 mg / mL.

[0233] The results for SEQ ID NO: 1 are shown in FIGS. 10A-10E. Based on the in vivo data presented above, SEQ ID NO: 1 was expected to inhibit cell proliferation in vitro. Despite these expectations, FIGS. 10A-10E suggest that SEQ ID NO: 1 did not inhibit cell growth in vitro. Without wishing to be bound by a particular theory, the conditions used in this example to plate the various cell types (which are different from the conditions used for the bovine collagen data presented in FIGS. 9A-9F) are thought to have affected the ability of SEQ ID NO: 1 to inhibit cell proliferation.

[0234] Example 3: Application of a Therapeutic Biomaterial Containing a Recombinant Collagen Fragment T-HEp3 cells expressing the CDK2 biosensor are injected orthotopically into mice. At the time of tumor resection, a therapeutic biomaterial filled with recombinant collagen fragments (or a control filled with DPBS) is applied to the wound area. Mice are monitored for recurrence of tumor cells after resection of the primary tumor and compared to the control group. In vivo imaging is used to visualize cancer cells. Cells arrested in G0 are determined by nuclear localization of the CDK2 sensor.

[0235] Example 4: Application of a Therapeutic Biomaterial Containing Recombinant Collagen Fragments T-HEp3 cells expressing the CDK2 biosensor are injected orthotopically into mice. The tumor is resected and sutured. After the skin has healed and the sutures are removed, a scar is visible. A therapeutic biomaterial filled with recombinant collagen fragments (or a control filled with DPBS) is applied to the scar. Mice are monitored for recurrence of tumor cells at the scar site and compared to the control group. In vivo imaging is used to visualize cancer cells. Cells arrested in G0 are determined by nuclear localization of the CDK2 sensor.

[0236] Example 5: Application of a Therapeutic Biomaterial Containing Recombinant Collagen Fragments T-HEp3 cells expressing the CDK2 biosensor are injected orthotopically into mice. The tumor is resected and sutured. A therapeutic biomaterial filled with recombinant collagen fragments (or a control filled with DPBS) is applied as a wound dressing immediately after surgery. Mice are monitored for recurrence of tumor cells after removal of the wound dressing and compared to the control group. In vivo imaging is used to visualize cancer cells. Cells arrested in G0 are determined by nuclear localization of the CDK2 sensor.

[0237] Example 6: Application of a Therapeutic Biomaterial Containing Recombinant Collagen Fragments T-HEp3 cells expressing the CDK2 biosensor are injected orthotopically into mice. The tumor is excised, and a therapeutic biomaterial filled with recombinant collagen fragments (or a control filled with DPBS) is applied inside the excision site and left there while applying sutures. The mice are monitored for recurrence of tumor cells after suture removal and compared to the control group. In vivo imaging is used to look for cancer cells. Cells arrested in G0 are determined by nuclear localization of the CDK2 sensor.

[0238] Examples 7 - 68 - Investigation of Therapeutic Biomaterials For use as the therapeutic biomaterials described herein, mixtures of HA and collagen, as well as mixtures of various other additional polymers and collagen, were investigated.

[0239] Example 7 Polyethylene oxide (PEO) with a molecular weight of 400 kDa was purchased from Sigma (Sigma - Aldrich, St. Louis, MO) ("Polymer 1"). A recombinant collagen fragment with a molecular weight of 50 kDa having SEQ ID NO: 1 was prepared in yeast as described in International Publication No. PCT / US2022 / 027016, which is hereby incorporated by reference in its entirety ("Polymer 2"). A 5 wt% polyethylene oxide (PEO) solution was prepared by dissolving 2.5 g of PEO in 47.5 ml of deionized (DI) water and mixing at 400 RPM for 16 hours on a stirring plate. A 5% (w / v) solution containing the recombinant collagen fragment having the amino acid sequence of SEQ ID NO: 1 was generated by dissolving 5 grams of the 50 kDa protein in 100 ml of water. In a 50 mL conical tube, the two solutions were mixed at a 1:1 (w / w) ratio and mixed at 10 RPM for 16 hours. The conical tube was placed vertically on a rack overnight, after which the solution stability was evaluated. The final solution was turbid, slowly phase - separated, and white or yellowish aggregates settled to the bottom of the tube.

[0240] Example 8 A mixture containing polyethylene oxide (PEO) and a recombinant collagen fragment having a molecular weight of 50 kDa with SEQ ID NO: 1 was prepared, dried, and visually examined. PEO having a molecular weight of 100 kDa was purchased from Sigma ("Polymer 1"). The rest of the method was the same as described in Example 7. The final solution was clear. The final solution phase-separated, with a turbid solution at the top and a slightly yellowish turbid solution at the bottom.

[0241] Example 9 A mixture of the first polyvinylpyrrolidone (PVP) and collagen (a recombinant collagen fragment having a molecular weight of 50 kDa with SEQ ID NO: 1) was prepared, dried, and visually examined. Example 9 was prepared using the same method as described in Example 7, except that Polymer 1 was replaced with polyvinylpyrrolidone (Sigma; molecular weight 10 kDa). The final solution was clear. Approximately 13 grams (g) of the solution was poured into a 100 ml polytetrafluoroethylene (PTFE) evaporating dish and dried overnight at 45°C. The dried film was visually clear.

[0242] This example demonstrates that a solution containing 10 kDa PVP and a recombinant collagen fragment having a molecular weight of 50 kDa with SEQ ID NO: 1 is completely miscible and can be prepared as a film or article.

[0243] Example 10 A mixture of the second polyvinylpyrrolidone (PVP) and collagen (a recombinant collagen fragment having a molecular weight of 50 kDa with SEQ ID NO: 1) was prepared, dried, and visually examined. Example 10 was prepared using the method described in Example 7, except that Polymer 1 was replaced with polyvinylpyrrolidone (Sigma; molecular weight 40 kDa). The final solution was clear. A film was prepared using the method described in Example 9. The dried film was visually clear.

[0244] This example demonstrates that a solution containing 40 kDa PVP and a recombinant collagen fragment with a molecular weight of 50 kDa having SEQ ID NO: 1 is completely miscible and can be prepared as a film or an article.

[0245] Example 11 A mixture of a third polyvinylpyrrolidone (PVP) and collagen (a recombinant collagen fragment having a molecular weight of 50 kDa having SEQ ID NO: 1) was prepared, dried, and visually examined. Example 11 was prepared using the method described in Example 7, except that Polymer 1 was replaced with polyvinylpyrrolidone (Sigma; molecular weight 360 kDa). The final solution was clear. A film was prepared using the same method as in Example 9. The dried film was visually transparent.

[0246] This example demonstrates that a solution containing 360 kDa PVP and a recombinant collagen fragment with a molecular weight of 50 kDa having SEQ ID NO: 1 is completely miscible and can be prepared as a film.

[0247] Example 12 A mixture of a first chitosan and collagen (a recombinant collagen fragment having a molecular weight of 50 kDa having SEQ ID NO: 1) was prepared and visually examined. Example 12 was prepared using the method described in Example 7, except that Polymer 1 was replaced with chitosan (molecular weight range 50 kDa - 190 kDa; Sigma). To completely solubilize the solution during mixing, 0.287 ml of acetic acid from Sigma was used to adjust the pH of the chitosan solution to pH 4. The final solution phase-separated into two layers. The viscosity was visually determined by examining the difference between the two layers. The upper layer had a lower viscosity and was more transparent, while the bottom layer had a higher viscosity and was more turbid. The viscosity was visually determined by examining the difference between the two layers.

[0248] Example 13 A mixture of polyethylene glycol and collagen (a recombinant collagen fragment having a molecular weight of 50 kDa with SEQ ID NO: 1) was prepared, dried, and visually examined. Example 14 was prepared using the method described in Example 7, except that Polymer 1 was replaced with polyethylene glycol (Sigma with a molecular weight of 35 kDa). The final solution phase-separated, with a clear and clarified solution at the top and a clear and slightly yellow solution at the bottom.

[0249] Example 14 A mixture of the first alginic acid and collagen (a recombinant collagen fragment having a molecular weight of 50 kDa with SEQ ID NO: 1) was prepared, dried, and visually examined. Example 14 was prepared using the method described in Example 7, except that Polymer 1 was replaced with a medium-viscosity sodium alginate salt (Sigma) from brown algae. The viscosity of the 2% sodium alginate salt in water at 25 °C was 2,000 cps or more. 2.5 g of sodium alginate salt was dissolved in 97.5 ml of deionized ("DI") water, and a 2.5 wt% alginic acid solution was prepared by mixing at 400 RPM for 24 hours on a stirring plate due to the high viscosity of the solution. The viscosity was visually determined by examining the difference between the two layers. In a 50 ml conical tube, the two solutions were mixed in a 1:1 (v / v) ratio and mixed at 10 RPM for 16 hours. The conical tube was placed vertically on a rack overnight, and then the solution stability was evaluated. The final solution was clear. A film was prepared using the method described in Example 9. The dried film was visually cloudy and uniformly dispersed and cloudy throughout the film.

[0250] Example 15 A mixture of the second alginic acid and collagen (a recombinant collagen fragment having a molecular weight of 50 kDa with SEQ ID NO: 1) was prepared, dried, and visually examined. Example 15 was prepared using the method described in Example 7, except that Polymer 1 was replaced with a low-viscosity sodium alginate salt (Sigma) from brown algae. The viscosity of 1% sodium alginate salt in water at 25 °C was about 4 - 12 cps. Both the sodium alginate salt and the collagen stock solution were at 5 wt%. In a 50 ml conical tube, the two solutions were mixed in a 1:1 (v / v) ratio and mixed at 10 RPM for 16 hours. The final solution was transparent. A film was prepared using the method described in Example 9. The dried film was visually turbid and was uniformly dispersed and turbid throughout the film.

[0251] Example 16 A mixture of polyacrylamide and collagen (a recombinant collagen fragment having a molecular weight of 50 kDa with SEQ ID NO: 1) was prepared, dried, and visually examined. Example 16 was prepared using the method described in Example 7, except that Polymer 1 was replaced with polyacrylamide (molecular weight range of 5,000 kDa - 6,000 kDa; Sigma). The rest of the method was the same as that described in Example 7. The final solution was transparent. A film was prepared using the same method described in Example 9. The dried film was visually transparent.

[0252] This example demonstrates that a solution containing polyacrylamide in the range of 5,000 kDa - 6,000 kDa and a recombinant collagen fragment having a molecular weight of 50 kDa with SEQ ID NO: 1 is miscible and can be prepared as a film or an article.

[0253] Example 17 A mixture of polyacrylamide and collagen (a recombinant collagen fragment having a molecular weight of 50 kDa with SEQ ID NO: 1) was prepared, dried, and visually examined. Example 17 was prepared using the method described in Example 7, except that Polymer 1 was replaced with polyacrylamide (molecular weight 40 kDa; Sigma). The rest of the method was the same as described in Example 7. The final solution was clear. A film was prepared using the method described in Example 9, except that an aluminum weighing pan was used. The dried film was visually transparent.

[0254] This example demonstrates that a solution containing 40 kDa polyacrylamide and a recombinant collagen fragment having a molecular weight of 50 kDa with SEQ ID NO: 1 is miscible and can be prepared as a film or an article.

[0255] Example 18 A mixture of a first polyvinyl alcohol (PVA) and collagen (a recombinant collagen fragment having a molecular weight of 50 kDa with SEQ ID NO: 1) was prepared and visually examined. Example 18 was prepared using the method described in Example 7, except that Polymer 1 was replaced with PVA (molecular weight range 89 kDa - 98 kDa; Sigma). A 4 wt% PVA solution was prepared by dissolving 2 g of PVA in 48 ml of DI water and mixing at 400 RPM for 16 hours on a stirring plate. In a 50 ml conical tube, the two solutions were mixed in a 1:1 (w / w) ratio and mixed at 10 RPM for 16 hours. The final solution phase-separated into two layers. The upper layer was turbid and a large amount of white powdery aggregates settled at the bottom.

[0256] Example 19 A mixture of a second PVA and collagen (a recombinant collagen fragment having a molecular weight of 50 kDa with SEQ ID NO: 1) was prepared and visually examined. Example 19 was prepared using the method described in Example 18, except that Polymer 1 was replaced with a 4% PVA (w / v) solution (unknown molecular weight; Fisher Scientific). The rest of the method was the same as that described in Example 18. The final solution phase-separated into two layers. The viscosity was visually determined by examining the difference between the two layers. The upper layer had a lower viscosity and was more turbid with small suspended particles. The bottom layer had a higher viscosity and was transparent.

[0257] Example 20 A mixture of a first hyaluronic acid (HA) and collagen (a recombinant collagen fragment having a molecular weight of 50 kDa with SEQ ID NO: 1) was prepared, dried, and visually examined. Example 20 was prepared using the method described in Example 7, except that Polymer 1 was replaced with hyaluronic acid (HA) (molecular weight of 1,000 kDa purchased from Pure Health Botanicals (Saint Charles, IL)). 5 g of HA was dissolved in 195 ml of DI water, and a 2.5 wt% HA solution was prepared by mixing at 400 RPM for 40 hours using a high-shear impeller due to the high viscosity of the solution. A 5% (w / v) collagen solution containing the recombinant collagen fragment of SEQ ID NO: 1 was produced as described in Example 7. In a 50 ml conical tube, the HA solution and the collagen solution were mixed at a ratio of 2:1 (w / w) and mixed at 10 RPM for 16 hours to maintain a dry mass ratio of 1:1. The final solution was transparent. A film was prepared using the method described in Example 9. The dried film was visually turbid and uniformly dispersed and turbid throughout the film.

[0258] Example 21 A mixture of a second hyaluronic acid (HA) and collagen (a recombinant collagen fragment having a molecular weight of 50 kDa with SEQ ID NO: 1) was prepared, dried, and visually examined. Example 21 was prepared using the method of Example 7, except that Polymer 1 was replaced with a non-animal-based HA (molecular weight of 50 kDa; Pure Health Botanicals (Saint Charles, IL) and DSM (Kaiseraugst, Switzerland)). A 5 wt% HA solution was prepared by dissolving 5 g of HA in 95 ml of DI water and mixing at 400 RPM for 16 hours on a stirring plate. A 5% (w / v) collagen solution containing the recombinant collagen fragment of SEQ ID NO: 1 was produced as described in Example 7. In a 50 ml conical tube, the two solutions were mixed at a 1:1 (w / w) ratio and mixed at 10 RPM for 16 hours. The final solution was clear. A film was prepared using the method described in Example 9. The dried film was visually cloudy and was uniformly dispersed and cloudy throughout the film. Additionally, when an aliquot of the solution mixture of HA and collagen was placed in a refrigerator at 4 °C overnight, the solution remained clear.

[0259] Example 22 A mixture of a first gelatin and HA was prepared and visually examined. Example 22 was prepared using the method of Example 21, except that Polymer 2 was replaced with a low molecular weight beef gelatin dietary supplement (Great Lakes Wellness, Grayslake, IL). A 2.2 wt% gelatin solution was prepared by dissolving 1 g of gelatin in 45 ml of 0.01 N HCl and mixing at 400 RPM for 16 hours using a high shear impeller due to the high viscosity of the solution. A 5 wt% 50 kDa HA solution was prepared using the method described in Example 21. In a 50 ml conical tube, the HA solution and the collagen solution were mixed at a 1:2.4 (w / w) ratio and mixed at 10 RPM for 16 hours to maintain a 1:1 dry mass ratio. The final solution phase-separated into two layers. The viscosity was visually determined by examining the difference between the two layers. The upper layer had a lower viscosity and was more transparent. The bottom layer had a higher viscosity and was more cloudy.

[0260] Example 23 A mixture of the second gelatin and HA was prepared and visually examined. Example 23 was prepared using the method described in Example 22, except that Polymer 2 was replaced with gelatin from porcine skin (molecular weight of 300 kDa; Sigma). 0.5 g of gelatin was dissolved in 45 ml of 0.01 N HCl, and a 1.1 wt% gelatin solution was prepared by mixing at 400 RPM for 16 hours using a high-shear impeller due to the high viscosity of the solution. A 5 wt% 50 kDa HA solution was prepared using the method described in Example 21. In a 50 ml conical tube, the HA solution and the collagen solution were mixed at a ratio of 1:4.5 (w / w) and mixed at 10 RPM for 16 hours to maintain a dry mass ratio of 1:1. The final solution phase-separated, with a large number of white aggregates settling to the bottom and scattered white aggregates suspended or floating on top.

[0261] Example 24 A mixture of HA and type I collagen was prepared and visually examined. Example 24 was prepared using the method described in Example 21, except that Polymer 2 was replaced with type I collagen (molecular weight of 414 kDa; Sigma). 0.3 g of type I collagen was dissolved in 45 ml of 0.01 N HCl, and a 0.7 wt% type I collagen solution was prepared by mixing at 400 RPM for 16 hours using a high-shear impeller due to the high viscosity of the solution. A 5 wt% 50 kDa HA solution was prepared using the method described in Example 21. In a 50 ml Falcon conical tube, the HA solution and the type I collagen solution were mixed together at a ratio of 1:7 (w / w) and mixed at 10 RPM for 16 hours using a Hula mixer to maintain a dry mass ratio of 1:1. The final solution phase-separated, with a large number of white fibrils settling to the bottom and scattered white fibrils suspended or floating on top.

[0262] Example 25 A mixture of cellulase and HA was prepared and visually examined. Example 25 was prepared using the method described in Example 21, except that Polymer 2 was replaced with cellulase (Sunson Industry Group Co., Ltd.). A 5% (w / v) cellulase solution was prepared by dissolving 2.5 g of cellulase in 47.5 ml of DI water and mixing at 400 RPM for 16 hours on a stirring plate. A 5 wt% 50 kDa HA solution was prepared using the same method described in Example 21. In a 50 ml conical tube, the two solutions were mixed together at a 1:1 (w / w) ratio and mixed at 10 RPM for 16 hours. The final solution was turbid and a yellow powder settled to the bottom.

[0263] Example 26 A mixture of HA, chitosan, and collagen (recombinant collagen fragment having a molecular weight of 50 kDa with SEQ ID NO: 1) was prepared and visually examined. Example 26 was prepared using the method described in Example 21, except that the chitosan used in Example 12 was added as Polymer 3. A 5 wt% 50 kDa HA solution was prepared by dissolving 5 g of HA in 95 ml of DI water and mixing at 400 RPM for 16 hours on a stirring plate. A 5% (w / v) collagen solution containing the recombinant collagen fragment of SEQ ID NO: 1 was generated by dissolving 5 grams of the 50 kDa protein in 100 ml of water as described in Example 7. A 5 wt% chitosan solution was prepared by dissolving 2.5 g of chitosan in 47.5 ml of DI water and mixing at 400 RPM for 16 hours on a stirring plate. The pH of the chitosan solution was adjusted to pH 4 using 0.287 ml of acetic acid from Sigma to completely solubilize the solution during mixing. In a 50 ml conical tube, the three solutions were mixed at a 1:1:1 (w / v) ratio and mixed at 10 RPM for 16 hours. The final solution phase-separated and large masses of yellow aggregates were suspended or floating in a low-viscosity medium.

[0264] Example 27 A mixture of hyaluronic acid and a recombinant collagen fragment having a molecular weight of 50 kDa with SEQ ID NO: 1 (hydroxylated up to 22%) was prepared and visually examined. The recombinant collagen fragment was hydroxylated by the process described in International Application No. 2021 / 163485, and the percentage of hydroxylation was measured by the process described in International Application No. 2021 / 163485 (the disclosure of which is hereby incorporated by reference in its entirety). Example 27 was prepared using the method of Example 21, except that Polymer 2 was replaced with 22% hydroxylated collagen having a molecular weight of 50 kDa. The 22% hydroxylated collagen solution had a solids content of 1.3% and a pH of about 6. A 5 wt% solution of 50 kDa HA (Polymer 1) was prepared by dissolving 5 g of HA in 95 ml of DI water and mixing at 400 RPM for 16 hours on a stirring plate. In a 15 ml Falcon tube, the two solutions were mixed in a 1:1 (w / w) ratio and mixed at 10 RPM for 16 hours. The final solution was turbid, indicating that the two polymers were not miscible in solution at this level of hydroxylation.

[0265] Example 28 A mixture of hyaluronic acid and a recombinant collagen fragment having a molecular weight of 50 kDa with SEQ ID NO: 1 (hydroxylated up to 44%) was prepared and visually examined. The recombinant collagen fragment was hydroxylated by the process described in International Application No. 2021 / 163485, and the percentage of hydroxylation was measured by the process described in International Application No. 2021 / 163485 (the entire disclosure of which is incorporated herein by reference). Example 28 was prepared using the same method as described in Example 27, except that 44% hydroxylated collagen having a molecular weight of 50 kDa was used as Polymer 2. The 44% hydroxylated collagen solution had a solids content of 1.3% and a pH of about 6. A 5 wt% 50 kDa HA solution (Polymer 1) was prepared by dissolving 5 g of HA in 95 ml of DI water and mixing at 400 RPM for 16 hours on a stirring plate. In a 15 ml conical tube, the two solutions were mixed in a 1:1 (w / w) ratio and mixed at 10 RPM for 16 hours. The final solution was turbid, slowly phase-separated, and white aggregates settled to the bottom of the tube.

[0266] Example 29 A mixture of a biphasic crosslinked hyaluronic acid (including crosslinked HA and non-crosslinked HA) and collagen (a recombinant collagen fragment having a molecular weight of 50 kDa with SEQ ID NO: 1) was prepared and visually examined. Example 29 was prepared using the method described in Example 21, except that Polymer 1 was replaced with biphasic crosslinked HA (particle size of 1.25 - 2 mm; Beijing Mengbring Bio-Sci-Tec.Co.,Ltd.). A 2.4% (w / v) HA solution was used. A solution of 20 wt% recombinant collagen fragment of SEQ ID NO: 1 (Polymer 2) was prepared by dissolving 2 g of 50 kDa recombinant collagen in 18 ml of DI water and mixing at 10 RPM for 4 hours. In a 50 ml conical tube, the two solutions were mixed together in a 1:1 (w / w) ratio and mixed at 10 RPM for 16 hours. The mixed solution was visually transparent.

[0267] This example demonstrates that a solution containing biphasic cross-linked HA and a recombinant collagen fragment with a molecular weight of 50 kDa having SEQ ID NO: 1 is completely miscible.

[0268] Example 30 A mixture of monophasic cross-linked hyaluronic acid and collagen (a recombinant collagen fragment having a molecular weight of 50 kDa having SEQ ID NO: 1) was prepared and visually examined. Example 30 was prepared using the same method as Example 21, except that Polymer 1 was replaced with monophasic cross-linked HA (Bloomage Biotechnology Corp. (Jinan, Shandong, China)). A 2% (w / v) monophasic cross-linked HA solution was used. A 20 wt% collagen solution containing the recombinant collagen fragment of SEQ ID NO: 1 was prepared by dissolving 2 g of 50 kDa recombinant collagen in 18 ml of DI water and mixing at 10 RPM for 4 hours. In a 50 ml conical tube, the two solutions were mixed at a 1:1 (w / w) ratio and mixed at 10 RPM for 16 hours. The solution was visually clear.

[0269] This example demonstrates that a solution containing monophasic cross-linked HA and a recombinant collagen fragment with a molecular weight of 50 kDa having SEQ ID NO: 1 is completely miscible.

[0270] Example 31 A mixture of biphasic cross-linked hyaluronic acid (containing cross-linked HA and non-cross-linked HA) and collagen (a recombinant collagen fragment having a molecular weight of 50 kDa with SEQ ID NO: 1) was prepared and visually examined. Example 31 was prepared using the method described in Example 30, except that Polymer 1 was replaced with biphasic cross-linked HA (Restylane). A 2.0% (w / v) biphasic cross-linked HA solution was used. A solution of 40 wt% recombinant collagen fragment of SEQ ID NO: 1 was prepared by dissolving 4 g of 50 kDa recombinant collagen in 16 ml of DI water and mixing at 10 RPM for 4 hours. In a 1 ml Eppendorf tube with a pipette, the two solutions were mixed at ratios of HA:collagen of 1:3, 1:1, 10:3, and 100:3 (w / w) and mixed at 10 RPM for 16 hours. The solutions were visually clear.

[0271] This example demonstrates that a solution containing biphasic cross-linked hyaluronic acid and a recombinant collagen fragment having a molecular weight of 50 kDa with SEQ ID NO: 1 is completely miscible.

[0272] Example 32 A mixture of 5% hyaluronic acid and collagen (a recombinant collagen fragment having a molecular weight of 50 kDa with SEQ ID NO: 1) was prepared and visually examined. Example 32 was prepared using the method described in Example 21, except that the pH of the 5 wt% 50 kDa HA solution was adjusted to pH 5.2 - 7.2 with sodium hydroxide before the addition of the collagen solution containing the recombinant collagen fragment of SEQ ID NO: 1. In a 50 ml tube, the two solutions were mixed together at a ratio of 1:1 (w / w) and mixed at 10 RPM for 16 hours. The solution was visually clear. A film was prepared using the method of Example 9. The dried film was visually cloudy and was uniformly dispersed and cloudy throughout the film.

[0273] This example demonstrates that a solution containing 5% hyaluronic acid at pH 5.2 - 7.2 and a recombinant collagen fragment having a molecular weight of 50 kDa with SEQ ID NO: 1 is completely miscible.

[0274] Example 33 Mixtures of 5% hyaluronic acid and collagen (a recombinant collagen fragment having a molecular weight of 50 kDa with SEQ ID NO: 1) were prepared at different pHs and visually examined. Example 33 was prepared using the method described in Example 21, except that the pH of the 5 wt% 50 kDa HA solution was adjusted to pH 9, 10, 11, and 12 with sodium hydroxide prior to the addition of the recombinant collagen fragment of SEQ ID NO: 1. In a 20 ml glass vial, the two solutions were mixed at a 1:1 (w / w) ratio and mixed for 30 minutes at 400 RPM using a stir bar due to the low viscosity of the basic solution. Then, while mixing at 400 RPM using a stir bar, the solution was readjusted to pH 7 ± 1 with concentrated hydrochloric acid. The solution was visually clear before and after adjusting the pH to 7 ± 1.

[0275] This example demonstrates that solutions containing 5% hyaluronic acid at pH 9, 10, 11, 12 and a recombinant collagen fragment having a molecular weight of 50 kDa with SEQ ID NO: 1 are completely miscible.

[0276] Example 34 A mixture of hyaluronic acid and collagen was prepared and visually examined. Example 34 was prepared using the method of Example 21, except that Polymer 1 was replaced with a 2% HumaColl21® solution (Geltor). Using the method described in Example 21, a 2 wt% 50 kDa HA solution was prepared. In a 2 ml glass vial, the two solutions were mixed at a 1:1 (w / w) ratio and mixed on a stirring plate at 400 RPM for 1 hour. The final solution was visually clear.

[0277] This example demonstrates that solutions containing a 2% HumaColl21® solution and a 50 kDa recombinant collagen fragment with SEQ ID NO: 1 are completely miscible.

[0278] Example 35 Blend hyaluronic acid, full-length recombinant collagen, and a 50 kDa recombinant collagen fragment, mix with HA, and visually examine. Blend the full-length recombinant collagen and the 50 kDa recombinant collagen fragment and mix with HA. Visually examine the solution. Mix a blend of the 50 kDa recombinant collagen fragment and the full-length recombinant collagen and / or hydrolyzed recombinant collagen (or any combination of these three) with HA and visually examine. Then, mix blends of the full-length recombinant collagen and the 50 kDa recombinant collagen fragment, and blends of the full-length recombinant collagen and hydrolyzed full-length recombinant collagen, with HA. Visually examine the solution.

[0279] Example 36 A poly(2-ethyl-2-oxazoline) (PEOx) solution was mixed with a solution containing a 50 kDa molecular weight recombinant collagen fragment having SEQ ID NO: 1. Example 36 was prepared using the same method described in Example 7, except that Polymer 1 was replaced with PEOx (50 kDa molecular weight; Sigma) 1. The remainder of the method was the same as that described in Example 7. The final solution was visually clear, indicating that the polymer was miscible in the solution. Films were prepared using the same method as described in Example 9, except that they were in aluminum weighing pans. The dried films visually appeared to phase separate during drying.

[0280] Example 37 A polyethyleneimine (PEI) solution was mixed with a solution containing a recombinant collagen fragment having a molecular weight of 50 kDa and having SEQ ID NO: 1. Example 37 was prepared using the method described in Example 7, except that Polymer 1 was replaced with PEI (molecular weight of 100 kDa; Polysciences (Warrington, Pennsylvania)). A 5 wt% polyethyleneimine solution was prepared by dissolving 0.5 g of PEI in 9.5 g of Milli-Q water at 80 °C using a stir bar at 450 RPM for 3 hours or until the solution became completely clear. The solution was then cooled to room temperature and the pH was adjusted to 7 using 12N HCl. The remainder of the method was the same as that described in Example 7. The solution was clear, indicating that the polymer was miscible in the solution. Films were prepared using the method described in Example 3. The dried films were visually opaque.

[0281] Example 38 A mixture of sodium carboxymethylcellulose (NaCMC) and collagen (a recombinant collagen fragment having a molecular weight of 50 kDa and having SEQ ID NO: 1) was prepared, dried, and examined visually. Example 38 was prepared using the method described in Example 7, except that Polymer 1 was replaced with sodium carboxymethylcellulose (molecular weight of approximately 250 kDa, degree of substitution of 1.22; Sigma). A 5 wt% sodium carboxymethylcellulose solution was prepared by dissolving 0.5 g of sodium carboxymethylcellulose in 9.5 g of DI water and mixing at 5 RPM for 16 hours in a conical tube. In a 50 ml conical tube, the two solutions were mixed in a 1:1 (v / v) ratio and mixed at 10 RPM for 16 hours. The final solution was clear and slightly yellow, indicating that the solutions were miscible. Films were prepared using the method described in Example 9, except that an aluminum weighing pan was used. The dried films were visually cloudy and uniformly dispersed and cloudy throughout the film.

[0282] Example 39 A mixture of the second sodium carboxymethyl cellulose and collagen (a recombinant collagen fragment having a molecular weight of 50 kDa and having SEQ ID NO: 1) was prepared, dried, and visually examined. Example 39 was prepared using the same method as described in Example 7, except that Polymer 1 was replaced with sodium carboxymethyl cellulose (molecular weight of about 250 kDa, degree of substitution of 0.79; Sigma). A 2.5 wt% sodium carboxymethyl cellulose solution was prepared by dissolving 0.25 g of sodium carboxymethyl cellulose in 9.75 g of DI water and mixing at 5 RPM for 16 hours in a conical tube. A 2.5 wt% collagen solution containing a recombinant collagen fragment having the amino acid sequence of SEQ ID NO: 1 was prepared by dissolving 0.25 g of 50 kDa recombinant collagen in 9.75 g of DI water. In a 50 ml conical tube, the two solutions were mixed in a 1:1 (v / v) ratio and mixed at 10 RPM for 16 hours. The final solution was clear, indicating that the polymer was miscible in the solution. A film was prepared using the method described in Example 9, except that an aluminum weighing pan was used. The dried film was visually cloudy and uniformly dispersed and cloudy throughout the film.

[0283] Example 40 A mixture of the third sodium carboxymethyl cellulose and collagen (a recombinant collagen fragment having a molecular weight of 50 kDa and having SEQ ID NO: 1) was prepared, dried, and visually examined. Example 41 was prepared using the method described in Example 7, except that Polymer 1 was replaced with sodium carboxymethyl cellulose (high viscosity of 2,700 cps and degree of substitution of 0.87 in a 1% aqueous solution; Sigma). A 2.5 wt% sodium carboxymethyl cellulose solution was prepared by dissolving 0.25 g of sodium carboxymethyl cellulose in 9.75 g of DI water and mixing at 5 RPM for 16 hours in a conical tube. A 2.5 wt% solution containing the recombinant collagen fragment having the amino acid sequence of SEQ ID NO: 1 was produced by dissolving 0.25 g of the recombinant collagen fragment in 9.75 g of water. In a 50 ml conical tube, the two solutions were mixed at a 1:1 (v / v) ratio and mixed at 10 RPM for 16 hours. The final solution was clear, indicating that the polymer was miscible in the solution. A film was prepared using the method described in Example 9, except that an aluminum weighing pan was used. The dried film was visually cloudy and uniformly dispersed and cloudy throughout the film.

[0284] Example 41 (HA / SEQ ID NO: 1007) A mixture of a recombinant collagen fragment having the amino acid sequence of SEQ ID NO: 1007 and HA was prepared, dried, and visually examined. SEQ ID NO: 1007 is 98% identical to SEQ ID NO: 1. Example 41 was prepared using the method described in Example 21, except that Polymer 2 was replaced with a collagen polymer having the amino acid sequence of SEQ ID NO: 1007. A 5 wt% solution containing the recombinant collagen fragment having the amino acid sequence of SEQ ID NO: 1007 was made by dissolving 0.25 g of the recombinant collagen fragment in 4.75 g of water. A 5 wt% solution of 50 kDa HA (Polymer 1) was prepared by dissolving 0.25 g of 50 kDa HA in 4.75 g of water. In a 50 ml conical tube, the two solutions were mixed at a 1:1 (w / w) ratio and mixed at 10 RPM for 16 hours. The conical tube was placed vertically on a rack overnight to evaluate solution stability. The final solution was clear. A film was prepared using the method described in Example 9, except that an aluminum pan was used. The dried film was visually cloudy and uniformly dispersed and cloudy throughout the film.

[0285] Example 42 (HA / SEQ ID NO: 1008) A mixture of a recombinant collagen fragment having the amino acid sequence of SEQ ID NO: 1008 and HA was prepared, dried, and visually examined. SEQ ID NO: 1008 is 50% identical to SEQ ID NO: 1. Example 42 was prepared using the method described in Example 21, except that Polymer 2 was replaced with a collagen polymer having the amino acid sequence of SEQ ID NO: 1008. A 5 wt% collagen solution containing a collagen polymer having the amino acid sequence of SEQ ID NO: 1008 was prepared by dissolving 0.25 g of the recombinant collagen fragment in 4.75 g of Milli-Q water (water purified using the Millipore Milli-Q lab water system; "MQ water") and purified through centrifugation at a relative centrifugal force (RCF) of 3214 for 1 hour. Using the same method described in Example 41, a 5 wt% solution of 50 kDa HA (Polymer 1) was prepared. In a 50 ml Falcon tube, the supernatant of the collagen solution and the HA solution were mixed at a ratio of 1:1 (w / w) and mixed at 10 RPM for 16 hours. The solution was clear, indicating that the two polymers were miscible in the solution. The dried film was visually cloudy.

[0286] Example 43 (HA / SEQ ID NO: 973) A mixture of full-length collagen and HA was prepared, dried, and visually examined. Example 43 was prepared using the method described in Example 41, except that Polymer 2 was replaced with a recombinant collagen fragment having a molecular weight of 50 kDa and having the amino acid sequence of SEQ ID NO: 973. A 5 wt% collagen solution containing the recombinant collagen fragment of SEQ ID NO: 973 was prepared by dissolving 0.25 g of the recombinant collagen in 4.75 g of MQ water. A 5 wt% solution of 50 kDa HA was prepared using the method described in Example 42. The rest of the method was the same as that described in Example 42. The solution was clear, indicating that the polymer was miscible in the solution. The dried film was visually cloudy and was uniformly dispersed and cloudy throughout the film.

[0287] Example 44 (chondroitin sulfate / collagen) A mixture of chondroitin sulfate and collagen (a recombinant collagen fragment having a molecular weight of 50 kDa with SEQ ID NO: 1) was prepared, dried, and visually examined. Example 44 was prepared using the method described in Example 7, except that Polymer 1 was replaced with chondroitin sulfate from bovine trachea (molecular weight of 50 kDa; EMD Millipore (Burlington, MA)). A 5 wt% chondroitin sulfate solution was prepared by dissolving 0.5 g of chondroitin sulfate in 9.5 g of MQ water. The remainder of the method was the same as that described in Example 7. The final solution was clear, indicating that the polymer was miscible in the solution. A film was prepared using the same method as described in Example 9, except that an aluminum weighing pan was used. The dried film was visually cloudy and uniformly dispersed and cloudy throughout the film.

[0288] Example 45 (AcHA / collagen) A mixture of acetylated hyaluronate (AcHA) and collagen (a recombinant collagen fragment having a molecular weight of 50 kDa with SEQ ID NO: 1) was prepared, dried, and visually examined. Example 45 was prepared using the method described in Example 21, except that Polymer 1 was replaced with sodium acetylated hyaluronate (Bloomage Biotechnology Corp., Ltd.). A 5 wt% acetylated hyaluronate (AcHA) solution was prepared by dissolving 0.25 g of AcHA in 4.75 g of MQ water. A 5 wt% solution containing the recombinant collagen fragment of SEQ ID NO: 1 was prepared by dissolving 0.25 g of collagen in 4.75 g of water. The remainder of the method was the same as that described in Example 21. The solution was clear, indicating that the polymer was miscible in the solution. A film was prepared using the same method as described in Example 9, except that an aluminum pan was used. The dried film was visually cloudy and uniformly dispersed and cloudy throughout the film.

[0289] Example 46 (ZnHA / collagen) A mixture of zinc hyaluronate (ZnHA) and collagen (a recombinant collagen fragment having a molecular weight of 50 kDa and having SEQ ID NO: 1) was prepared, dried, and visually examined. Example 46 was prepared using the method described in Example 21, except that polymer 1 was replaced with zinc hyaluronate (ZnHA) (molecular weight 170 kDa; Bloomage Biotechnology Corp., Ltd.). A 5 wt% zinc hyaluronate (ZnHA) solution was prepared by dissolving 0.25 g of ZnHA in 4.75 g of MQ water. A 5 wt% solution containing the recombinant collagen fragment of SEQ ID NO: 1 was prepared by dissolving 0.25 g of collagen in 4.75 g of water. The remainder of the method was the same as that described in Example 21. The solution was turbid and showed aggregation of particles. A film was prepared using the same method as described in Example 9, except that an aluminum pan was used. The dried film was transparent, indicating that the polymers were miscible.

[0290] Example 47 (HA / collagen salt doping study) A solution of hyaluronic acid and collagen (a recombinant collagen fragment having a molecular weight of 50 kDa and having SEQ ID NO: 1) was prepared, dried, and visually examined. Example 47 was prepared using the method described in Example 21, except that a solution containing the recombinant collagen fragment having a molecular weight of 50 kDa and having SEQ ID NO: 1 was prepared in an NaCl solution. A 5 wt% collagen solution was prepared by dissolving 4.75 g in 0.25 g in a 0.05 mM NaCl solution. A 5 wt% 50 kDa HA solution was prepared using the same method as described in Example 41. In a 50 ml conical tube, the two solutions were mixed in a 1:1 (w / w) ratio and mixed at 10 RPM for 4 hours. The solution was transparent, indicating that the polymer was miscible in a solution with a higher salt content. A film was prepared using the same method as described in Example 9, except that an aluminum pan was used. The dried film was turbid.

[0291] Example 48 (HA / Collagen Dialysis) A solution of hyaluronic acid and collagen (a recombinant collagen fragment having a molecular weight of 50 kDa with SEQ ID NO: 1) was prepared, dried, and visually examined. Example 48 was prepared using the method described in Example 21, except that the solution of hyaluronic acid and collagen was dialyzed. A 5 wt% collagen solution containing the recombinant collagen fragment of SEQ ID NO: 1 was prepared by dissolving 0.25 g of recombinant collagen in 4.75 g of MQ water. A 5 wt% 50 kDa HA solution was prepared by dissolving 0.25 g of hyaluronic acid powder in 4.95 g of MQ water. In a 50 ml conical tube, the two solutions were mixed at a 1:1 (w / w) ratio and mixed at 10 RPM for 4 hours. Then, 9 g of the combined solution was loaded onto a 10 ml volume 3.5 - 5 kDa molecular weight cut-off (MWCO) membrane. A dialysis fluid reservoir was prepared with a Nalgene bottle containing 500 ml of MQ water and a stir bar for gently rotating the surrounding solution continuously. The membrane containing the solution was submerged in the dialysis fluid reservoir and dialyzed at room temperature for 16 hours. The solution was pipetted out of the membrane and transferred to a 50 ml conical tube. The solution was clear, indicating that the polymer was miscible in a solution with a lower salt content. A film was prepared using the same method as described in Example 9, except using an aluminum pan. The dried film was visually cloudy and was uniformly dispersed and cloudy throughout the film.

[0292] Example 49 (HA and Hydrolyzed Recombinant Collagen Fragment - 5 mg / mL Papain) A solution of hyaluronic acid and hydrolyzed collagen (hydrolyzed fragment of a recombinant collagen fragment having a molecular weight of 50 kDa with SEQ ID NO: 1) was prepared, dried, and visually examined. A 5 wt% solution containing a recombinant collagen fragment having the amino acid sequence of SEQ ID NO: 1 was prepared by dissolving 0.25 g of collagen in 4.75 g of water. A 10 wt% papain enzyme solution was prepared by dissolving 100 mg of papain (200 TU / mg, supplied by BIO-CAT) in 900 μl of water. 250 μL of the 10 wt% papain enzyme solution was added to 4.75 ml of the 5 wt% recombinant collagen solution, and incubated at 60 °C for 2 hours and then at 90 °C for 10 minutes for enzymatic hydrolysis to inactivate the enzyme activity, thereby preparing a hydrolyzed recombinant fragment solution. Thereafter, the hydrolyzed solution was centrifuged at a relative centrifugal force (RCF) of 3214 for 20 minutes. The supernatant solution was collected and mixed with the following HA solution. A solution of 5 wt% recombinant collagen fragment and HA was prepared by dissolving 0.25 g of 50 kDa HA in 4.75 g of water. In a 50 ml conical tube, the two solutions were mixed at a ratio of 1:1 (w / w) and mixed at 5 RPM for 2 hours. The conical tube was placed vertically on a rack overnight to evaluate the solution stability. The final solution was transparent. Films were prepared using the method described in Example 9, except that a silicone mold was used. The dried film was visually transparent, indicating that the polymers were completely miscible.

[0293] Example 50 (PVP and Hydrolyzed Recombinant Collagen Fragment - 5 mg / mL Papain) A solution of polyvinylpyrrolidone and hydrolyzed recombinant collagen (hydrolyzed fragment of a recombinant collagen fragment having a molecular weight of 50 kDa with SEQ ID NO: 1) was prepared, dried, and visually examined. Example 50 was prepared using the method described in Example 49, except that Polymer 1 was replaced with polyvinylpyrrolidone (molecular weight 40 kDa; Sigma). A 5 wt% 40 kDa PVP solution was prepared by dissolving 0.25 g of 40 kDa PVP in 4.75 g of water. A hydrolyzed recombinant collagen solution was prepared using the method described in Example 43. In a 50 ml conical tube, the two solutions were mixed at a 1:1 (w / w) ratio and mixed at 5 RPM for 2 hours. The conical tube was placed vertically on a rack overnight to evaluate solution stability. The final solution was clear. A film was prepared using the method described in Example 9, except that a silicone mold was used. The dried film was visually clear, indicating that the polymer was miscible in both the solution and the solid.

[0294] Example 51 (HA, and blend of recombinant collagen fragment and hydrolyzed recombinant collagen fragment - 0.1 mg / mL papain) A solution of hyaluronic acid, a recombinant collagen fragment having a molecular weight of 50 kDa with SEQ ID NO: 1, and a partially hydrolyzed recombinant collagen fragment was prepared, dried, and visually examined. A 5 wt% solution containing the recombinant collagen fragment having the amino acid sequence of SEQ ID NO: 1 was prepared by dissolving 0.25 g of the recombinant collagen fragment in 4.75 g of water. A blend of the recombinant collagen fragment and the hydrolyzed recombinant collagen fragment solution was prepared by partially hydrolyzing the 5 wt% collagen solution. A 1 wt% papain enzyme solution was prepared by dissolving 10 mg of papain (200 TU / mg, supplied by BIO-CAT) in 990 μl of water. 50 μL of the 1 wt% papain enzyme solution was added to 4.95 ml of the 5 wt% recombinant collagen solution and incubated at 60 °C for 2 hours and then at 90 °C for 10 minutes to inactivate the enzyme activity, thereby preparing a solution of the partially hydrolyzed recombinant fragment (Polymer 2). A 5 wt% 50 kDa HA solution was prepared (Polymer 1) by dissolving 0.25 g of 50 kDa HA in 4.75 g of water. In a 50 ml conical tube, the two solutions were mixed at a 1:1 (w / w) ratio and mixed at 5 RPM for 2 hours. The conical tube was placed vertically on a rack overnight to evaluate the solution stability. The final solution was clear, indicating that the polymer was miscible in the solution. Films were prepared using the method described in Example 9, except that a silicone mold was used. The dried film was visually cloudy and uniformly dispersed and cloudy throughout the film.

[0295] Example 52 (PVP, and a blend of recombinant collagen fragment and hydrolyzed recombinant collagen fragment - 0.1 mg / mL papain) A solution of polyvinylpyrrolidone, a recombinant collagen fragment having a molecular weight of 50 kDa with SEQ ID NO: 1, and a partially hydrolyzed recombinant collagen fragment was prepared, dried, and visually examined. Example 52 was prepared using the method described in Example 51, except that Polymer 1 was replaced with polyvinylpyrrolidone (molecular weight 40 kDa; Sigma). A 5 wt% 40 kDa PVP solution was prepared by dissolving 0.25 g of 40 kDa PVP in 4.75 g of water. Using the method described in Example 51, a blend of the recombinant collagen fragment and the hydrolyzed recombinant collagen fragment solution was prepared. In a 50 ml conical tube, the two solutions were mixed at a 1:1 (w / w) ratio and mixed at 5 RPM for 2 hours. The conical tube was placed vertically on a rack overnight to evaluate solution stability. The final solution was clear. A film was prepared using the method described in Example 9, except that an aluminum weighing pan was used. The dried film was visually clear, indicating that the polymer was miscible in both the solution and the solid.

[0296] Example 53 (Biphasic HA, and a blend of recombinant collagen fragment and hydrolyzed recombinant collagen fragment - 0.1 mg / mL papain) A solution was prepared and visually examined of a blend of a biphasic cross-linked hyaluronic acid (containing cross-linked HA and non-cross-linked HA) and a recombinant collagen fragment having a molecular weight of 50 kDa with the sequence number 1 and a partially hydrolyzed recombinant collagen fragment. Example 53 was prepared using the method described in Example 51, except that Polymer 1 was replaced with 2.4% (w / v) biphasic cross-linked HA having a particle size of 0.10 - 0.25 mm (Shandong Runxin Biotechnology Co., Ltd., Qufu City, Shandong Province, China). Using the method described in Example 51, a solution containing a blend of a recombinant collagen fragment having a molecular weight of 50 kDa with the sequence number 1 and a hydrolyzed recombinant collagen fragment was prepared. In a 50 ml conical tube, the two solutions were mixed at a ratio of 1:1 (w / w), vortexed for 3 minutes, and mixed at 5 RPM for 3 hours. The conical tube was placed vertically on a rack overnight to evaluate solution stability. Due to the visible cross-linked HA particle texture, the solution was visually slightly turbid.

[0297] Example 54 (Sodium CMC, and a blend of a recombinant collagen fragment and a hydrolyzed recombinant collagen fragment - 0.1 mg / mL papain) A solution was prepared from sodium carboxymethylcellulose and a blend of a recombinant collagen fragment having a molecular weight of 50 kDa with SEQ ID NO: 1 and a partially hydrolyzed recombinant collagen fragment, dried, and visually examined. Example 54 was prepared using the method described in Example 51, except that Polymer 1 was replaced with sodium carboxymethylcellulose (molecular weight of approximately 250 kDa, degree of substitution of 1.22; Sigma). A 5 wt% sodium carboxymethylcellulose solution was prepared by dissolving 0.5 g of sodium carboxymethylcellulose in 9.5 g of DI water and mixing at 5 RPM for 16 hours in a conical tube. A blend of the recombinant collagen fragment and the hydrolyzed recombinant collagen fragment solution was prepared using the method described in Example 51. In a 50 ml conical tube, the two solutions were mixed at a 1:1 (w / w) ratio and mixed at 5 RPM for 2 hours. The conical tube was placed vertically on a rack overnight to evaluate solution stability. The final solution was clear, indicating that the polymer was miscible in the solution. A film was prepared using the method described in Example 9, except that an aluminum weighing pan was used. The dried film was visually cloudy and uniformly dispersed and cloudy throughout the film.

[0298] Example 55 (Sodium Carboxymethylcellulose and SEQ ID NO: 1007) A solution of sodium carboxymethyl cellulose and a collagen polymer having the amino acid sequence set forth in SEQ ID NO: 1007 was prepared, dried, and visually examined. SEQ ID NO: 1007 is 98% identical to SEQ ID NO: 1. A 5 wt% solution containing a recombinant collagen polymer having the amino acid sequence set forth in SEQ ID NO: 1007 was prepared by dissolving 0.5 g of the recombinant collagen fragment in 9.5 g of water. A 5 wt% solution of sodium carboxymethyl cellulose (molecular weight of about 250 kDa and degree of substitution of 1.22; Sigma) was prepared by dissolving 0.5 g of sodium carboxymethyl cellulose in 9.5 g of DI water and mixing at 5 RPM for 16 hours in a conical tube. In a 50 ml conical tube, the two solutions were mixed at a 1:1 (w / w) ratio and mixed at 5 RPM for 2 hours. The conical tube was placed vertically on a rack overnight to evaluate solution stability. The final solution was clear, indicating that the polymer was miscible in the solution. Films were prepared using the method described in Example 9, except that a silicone mold was used. The dried films were visually cloudy and uniformly dispersed and cloudy throughout the film.

[0299] Example 56 (PVP and SEQ ID NO: 1007) A solution of polyvinylpyrrolidone and a collagen polymer having the amino acid sequence set forth in SEQ ID NO: 1007 was prepared, dried, and visually examined. SEQ ID NO: 1007 is 98% identical to SEQ ID NO: 1. A 5 wt% solution containing a recombinant collagen fragment having the amino acid sequence set forth in SEQ ID NO: 1007 was prepared by dissolving 0.5 g of the recombinant collagen fragment in 9.5 g of water. A 5 wt% 40 kDa PVP solution was prepared by dissolving 0.25 g of 40 kDa PVP in 4.75 g of water. In a 50 ml conical tube, the two solutions were mixed at a 1:1 (w / w) ratio and mixed at 5 RPM for 2 hours. The conical tube was placed vertically on a rack overnight to evaluate solution stability. The final solution was clear. A film was prepared using the method described in Example 9, except that a silicone mold was used. The dried film was visually clear, indicating that the polymer was miscible in the solution in the solid state.

[0300] Example 57 (Biphasic HA and SEQ ID NO: 1007) A blend of a biphasic cross-linked hyaluronic acid (containing cross-linked HA and non-cross-linked HA) and a solution of a recombinant collagen fragment having the amino acid sequence set forth in SEQ ID NO: 1007 and a hydrolyzed recombinant collagen fragment was prepared and visually examined. SEQ ID NO: 1007 is 98% identical to SEQ ID NO: 1. 2.4% (w / v) biphasic cross-linked HA having a particle size of 0.10 - 0.25 mm purchased from Shandong Runxin Biotechnology Co., Ltd. (Qufu City, Shandong Province, China) was used as Polymer 1. A 5 wt% solution containing a recombinant collagen fragment having the amino acid sequence set forth in SEQ ID NO: 1007 was prepared by dissolving 0.5 g of the recombinant collagen fragment in 9.5 g of water. In a 50 ml conical tube, the two solutions were mixed at a ratio of 1:1 (w / w), vortexed for 3 minutes, and mixed at 5 RPM for 3 hours. The conical tube was placed vertically on a rack overnight to evaluate the solution stability. The solution was visually clear. Additionally, when the same conical tube was placed vertically on a rack in a refrigerator at 4°C overnight, the solution remained clear, indicating that the polymer was miscible in the solution.

[0301] Example 58 (HA:SEQ ID NO: 1 at different ratios) A mixture of hyaluronic acid (HA) and collagen (a recombinant collagen fragment having a molecular weight of 50 kDa with SEQ ID NO: 1) was prepared, dried, and visually examined. A 5 wt% 50 kDa HA solution was prepared by dissolving 2.5 g of HA in 47.5 g of DI water. A 5% (w / v) collagen solution containing the recombinant collagen fragment of SEQ ID NO: 1 was made by dissolving 2.5 g of the recombinant collagen fragment in 47.5 g of water. In a 50 ml conical tube, two solutions having a total solution weight of 10 g were mixed at HA:collagen ratios of 1:9, 1:3, 2:3, 1:1, 3:2, 3:1, and 9:1 (w / w) and mixed at 5 RPM for 3 hours. The conical tubes were placed vertically on a rack overnight to evaluate solution stability. The final solutions were clear, indicating that all ratios were miscible in solution. Films were prepared using the same method as described in Example 9, except using a silicone mold. The dried film containing HA:collagen in a ratio of 1:9 was visually clear. The dried film containing HA:collagen in a ratio of 1:3 was visually clear to slightly turbid. The dried films of the remaining ratios were visually turbid and were uniformly distributed and turbid throughout the film, indicating that miscibility in the solid state depends on the composition. Additionally, aliquots of these seven ratios of HA:collagen solution mixtures were placed in a refrigerator at 4°C overnight, and the solutions remained clear.

[0302] Example 59 (HA: 5% hydroxylation) A mixture of hyaluronic acid and a 5% hydroxylated recombinant collagen fragment having a molecular weight of 50 kDa with SEQ ID NO: 1 was prepared and visually examined. The recombinant collagen fragment was hydroxylated by the process described in International Application No. 2021 / 163485, and the percent hydroxylation was measured by the process described in International Application No. 2021 / 163485 (the disclosure of which is incorporated herein by reference in its entirety). Example 60 was prepared using the method described in Example 27, except that Polymer 2 was replaced with 5.41% hydroxylated collagen (molecular weight 50 kDa). A 2 wt% solution of 50 kDa HA (Polymer 1) was prepared by dissolving 0.2 g of HA in 9.8 g of MQ water. The 5% hydroxylated collagen had a solids content of 12% and a pH of about 6. The hydroxylated collagen solution was then diluted to 2 wt% with water and purified by centrifugation at 15,000 RPM for 10 minutes. In a 15 ml Falcon tube, the supernatant of the hydroxylated collagen solution and the HA solution were mixed in a 1:1 (w / w) ratio and mixed at 10 RPM for 16 hours. The solution was clear, indicating that the polymer was miscible in the solution. A film was prepared using the method described in Example 9, except that it was within a silicon mold. The dried film was visually cloudy.

[0303] Example 60 (HA: 12% hydroxylated) A mixture of hyaluronic acid and a 12% hydroxylated recombinant collagen fragment having a molecular weight of 50 kDa with SEQ ID NO: 1 was prepared and visually examined. The recombinant collagen fragment was hydroxylated by the process described in International Application No. 2021 / 163485, and the percent hydroxylation was measured by the process described in International Application No. 2021 / 163485 (the disclosure of which is hereby incorporated by reference in its entirety). Example 60 was prepared using the method described in Example 27, except that 12.47% hydroxylated collagen having a molecular weight of 50 kDa was used as Polymer 2. A 2 wt% solution of 50 kDa HA (Polymer 1) was prepared by dissolving 0.2 g of HA in 9.8 g of MQ water. The 12% hydroxylated collagen had a solids content of 3% and a pH of approximately 6. The hydroxylated collagen solution was then diluted to 2 wt% with water and purified by centrifugation at 15,000 RPM for 10 minutes. In a 15 ml Falcon tube, the supernatant of the hydroxylated collagen solution and the HA solution were in a 1:1 (w / w) ratio and mixed at 10 RPM for 16 hours. The solution was clear, indicating that the polymer was miscible in the solution. A film was prepared using the method described in Example 9, except that it was within a silicone mold. The dried film was visually cloudy.

[0304] Example 61 (5% hydroxylation in HA:HCl) A mixture of hyaluronic acid and a 5% hydroxylated recombinant collagen fragment having a molecular weight of 50 kDa with SEQ ID NO: 1 was prepared and visually examined. The recombinant collagen fragment was hydroxylated by the process described in International Application No. 2021 / 163485, and the percent hydroxylation was measured by the process described in International Application No. 2021 / 163485 (the disclosure of which is incorporated herein by reference in its entirety). Example 61 was prepared using the method described in Example 27, except that 5.41% hydroxylated collagen having a molecular weight of 50 kDa was used as Polymer 2. A 2 wt% 50 kDa HA solution (Polymer 1) was prepared by dissolving 0.2 g of HA in 9.8 g of MQ water. The 5% hydroxylated collagen had a solids content of 12% and a pH of about 6. The hydroxylated collagen solution was then diluted to 2 wt% with water and the pH was adjusted to about 2.2 using 12N HCl. The collagen solution was purified by centrifugation at 15,000 RPM for 10 minutes. In a 15 ml Falcon tube, the supernatant of the hydroxylated collagen solution and the HA solution were mixed in a 1:1 (w / w) ratio and mixed at 10 RPM for 16 hours. The solution was cloudy. The collagen and HA solutions were neutralized using 10N NaOH. The solution was clear. A film was prepared using the method described in Example 9, except that it was within a silicon mold. The dried film was visually cloudy.

[0305] Example 62 (12% Hydroxylated in HA:HCl) A mixture of hyaluronic acid and a 12% hydroxylated recombinant collagen fragment having a molecular weight of 50 kDa with SEQ ID NO: 1 was prepared and visually examined. The recombinant collagen fragment was hydroxylated by the process described in International Application No. 2021 / 163485, and the percent hydroxylation was measured by the process described in International Application No. 2021 / 163485 (the disclosure of which is hereby incorporated by reference in its entirety). Example 62 was prepared using the method described in Example 27, except that 12.47% hydroxylated collagen having a molecular weight of 50 kDa was used as Polymer 2. A 2 wt% solution of 50 kDa HA (Polymer 1) was prepared by dissolving 0.2 g of HA in 9.8 g of MQ water. The 12% hydroxylated collagen had a solids content of 3% and a pH of approximately 6. The hydroxylated collagen solution was then diluted to 2 wt% with water and the pH was adjusted to approximately 2.2 using 12N HCl. The collagen solution was purified by centrifugation at 15,000 rpm for 10 minutes. In a 15 ml Falcon tube, the supernatant of the hydroxylated collagen solution and the HA solution were mixed in a 1:1 (w / w) ratio and mixed at 10 RPM for 16 hours. The solution was cloudy. The collagen and HA solutions were neutralized using 10N NaOH. The solution was clear. A film was prepared using the method described in Example 9, except that it was within a silicon mold. The dried film was visually cloudy.

[0306] Example 63 (SEQ ID NO: 1 Foam Preparation) A stock aqueous solution of collagen (SEQ ID NO: 1) was prepared at a weight concentration of 16.7% (w / w). Baymedix FD103 and Baymedix® AD111 are each aqueous polyurethane dispersions. A stock solution of Baymedix (Bayer MaterialScience, Pittsburgh, PA) FD103:AD111 was prepared at a ratio of 70:30 based on the weight of the polyurethane dispersion. A stock aqueous solution of Rheolate208 (Elementis, London, UK) was prepared at a weight concentration of 10% (w / w). 9.16 g of the recombinant collagen stock solution and 11.50 g of the Baymedix FD103:AD111 stock solution were mixed in a 50 mL beaker using an overhead mixer. Then, 1.55 g of the Rheolate208 stock solution was added and mixed. Next, 0.72 g of ChemTex (Cumberland, RI) 2216, 0.65 g of ChemTex2317, and 0.03 g of ChemTex2243 were added and the resulting mixture was further mixed. The resulting mixture was mechanically foamed by vigorously mixing using an overhead mixer. After foaming, the foamed mixture was coated onto a glass plate with a 600 μm gap setting. The coating was dried at 75 °C for 30 minutes to obtain a porous foam sheet after drying.

[0307] Example 64 (Collagen Protein Release Kinetics) (A) The release kinetics of a solution of a recombinant collagen having the amino acid sequence of SEQ ID NO: 1 and (B) a formulation of a recombinant collagen having the amino acid sequence of SEQ ID NO: 1 and hyaluronic acid were investigated. The results are shown in FIGS. 11A, 11B, 12A, and 12B.

[0308] Analysis Standard curve: The three obtained absorbance values for each sample and dilution were averaged and subtracted from the blank (1×PBS + 0.5% sodium benzoate) value to obtain the mean absorbance value (MAV) from only the collagen in each sample / dilution. The MAVs of serial dilutions from 10 to 0.5 mg / mL were plotted against their respective concentrations. The same was done for dilutions from 0.08 to 0.02 mg / mL. A linear trend line was plotted for the range from 0.08 to 0.02 mg / mL. The equation y = 0.7033× - 0.0131 was obtained, and the 2 R value was 0.9936. This equation was used to calculate the protein concentrations of the test samples for both control (A) and formulation (B) from their respective MAVs (Figure 11A). The collagen mass per sample was calculated by multiplying the total solution volume present externally during the diffusion experiment (90 mL, 89.8 mL, 89.6 mL, or 89.4 mL).

[0309] The concentration was plotted against the time point to obtain the release trend. Two such trends were ultimately obtained, one for solution (A) and the other for formulation (B) (Figure 11B). As shown in the figure, the protein is gradually released from both solution A and formulation B containing HA. The rate at which the protein is released from formulation B appears to be slightly slower than from the solution without HA. This indicates a weak but measurable interaction between HA and collagen, most likely due to the miscibility of the solutions.

[0310] Micro BCA assay For the analysis using the Micro BCA assay, the Micro BCATM Protein Assay Kit protocol was referred to.

[0311] Analysis The three obtained absorbance values for each sample and dilution were averaged and subtracted from the blank (1×PBS + 0.5% sodium benzoate) value to obtain the mean absorbance value (MAV) from only the collagen in each sample / dilution. The standard deviation and error were calculated for each value.

[0312] The MAVs of serial dilutions from 10 to 0.5 mg / mL were plotted against their respective concentrations. The same was done for dilutions from 0.08 to 0.02 mg / mL. A linear trend line was plotted for the range from 0.08 to 0.02 mg / mL. The equation y = 13.312x + 0.0692 was obtained, and the R 2 value was 0.9918. Using this equation, the protein concentrations of the test samples for both control (A) and formulation (B) were calculated from their respective MAVs (Figure 12A). The collagen mass per sample was calculated by multiplying the total solution volume present externally during the diffusion experiment (90 mL, 89.8 mL, 89.6 mL, or 89.4 mL). The concentrations were plotted against time points to obtain the trend of the amount of protein released from the solution / formulation. Two such trends were finally obtained, one for solution (A) and the other for formulation (B) (Figure 12B). As shown, the protein diffused gradually from both solution (A) and formulation (B). The protein appeared to diffuse more slowly from formulation (B), indicating the attractive force between HA and collagen.

[0313] Example 65 (Fluorescent Labeling for Collagen Protein Release Kinetics) The release kinetics of the control solution (A) recombinant collagen having the amino acid sequence of SEQ ID NO: 1 and the formulation (B) formulation of recombinant collagen having the amino acid sequence of SEQ ID NO: 1 and hyaluronic acid were fluorescently labeled to display the release kinetic data. The results are shown in Figures 13A and 13B.

[0314] Analysis All dilutions of the standard curve and time points from the inside of (A) and (B) were pipetted onto a 96-well plate. Three technical replicates of 100 μL each were pipetted. The fluorescence intensity was measured by setting the excitation wavelength and emission wavelength to 680 nm and 702 nm, respectively.

[0315] Standard curve: The three intensity values obtained for each sample and diluent were averaged to obtain the mean intensity value (MIV). The MIVs of serial dilutions of 0.0625, 0.03125, 0.015625, 0.0078125, 0.00390625 mg / mL were plotted against their respective concentrations. A linear trend line was plotted (Figure 13A). The equation y = 72137x + 253.72 was obtained and the R2 value was 0.9967. This equation was used to calculate the protein concentrations of the test samples for both solution (A) and formulation (B) from their respective MIVs. The unlabeled collagen concentration was calculated by multiplying the labeled concentration value by 50. These concentrations were plotted against time points to obtain the trend of the amount of protein remaining in the solution / formulation (Figure 13B). As shown in Figure 13B, collagen diffuses gradually from both solution (A) and formulation (B). Collagen appears to diffuse more slowly from the formulation containing HA (formulation (B)), indicating an attractive force between HA and collagen.

[0316] Example 66 (Collagen Protein Release Kinetics / Crosslinked HA) (A) The release kinetics of a 1% solution of recombinant collagen having the amino acid sequence of SEQ ID NO: 1, and (B) a solution of a formulation of recombinant collagen having the amino acid sequence of SEQ ID NO: 1 and crosslinked hyaluronic acid were investigated. The results are shown in Figures 14A and 14B.

[0317] Analysis Three absorbance values for each sample and dilution were obtained, averaged, and subtracted from the blank (1×PBS + value) to obtain the mean absorbance value (MAV) from only the collagen in each sample / dilution. The standard deviation and error were calculated for each value.

[0318] The MAVs of serial dilutions of 0.2 - 0.01 mg / mL were plotted against their respective concentrations. A linear trend line was plotted for the range of 0.2 - 0.01 mg / mL (Figure 14A). The equation y = 13.947x was obtained and the R2 value was 0.9921. The trend line is in good agreement with the prediction and is suitable for use in converting absorbance values to concentrations for diffusion experiments.

[0319] Using this equation, the collagen concentration of the test samples of both the control (A) and the formulation (B) was calculated from their respective MAVs. The collagen mass per sample was calculated by multiplying the total solution volume present externally during the diffusion experiment (95 mL, 94 mL, 93 mL, or 92 mL).

[0320] The concentration was plotted against time points to obtain the trend of the amount of collagen released from the solution / formulation (Figure 14B). Two such trends were ultimately obtained, one for the collagen solution (A) and the other for the collagen / HA formulation (B). As shown in Figure 7B, the collagen release rate from the control solution (A) was the same as in the previous control example. However, the collagen was released much more slowly from the crosslinked HA / collagen formulation (B). For example, the amount of protein released from formulation (B) on day 8 took only an average of 3 days to diffuse from the control solution (A). After 19 days, the collagen was completely released from formulation (B).

[0321] Example 67 A mixture of polyethylene glycol (PEG) and collagen was prepared, dried, and visually examined. Example 67 was prepared using the method described in Example 7, except that Polymer 1 was replaced with polyethylene glycol (molecular weight 8 kDa; Sigma). The final solution was visually clear, indicating that the two polymers were miscible in the solution. A film was prepared using the method described in Example 9. The dried film separated into two phases, with small cracked pieces of a brittle opaque material surrounded by a ring of a brownish transparent material.

[0322] Example 68 A mixture of hyaluronic acid and collagen was prepared and visually examined. Example 68 was prepared using the method described in Example 20, except that Polymer 1 was replaced with HumaColl21® powder from Geltor. Using the method described in Example 20, a 2 wt% solution of 50 kDa HA was prepared. In a 15 ml conical tube, the two solutions were mixed together at a 1:1 (w / w) ratio and mixed on a stirring plate at 10 RPM for 16 hours. The final solution was visually clear. Films were prepared using the method described in Example 9, except that an aluminum weighing pan was used. The dried films were transparent with a milky white ring along the edges of the film, which was likely the preservative.

[0323] This example demonstrates that a solution containing HumaColl21® powder and 50 kDa HA is miscible.

[0324] Example 69 A series of in vivo experiments are conducted using the chick chorioallantoic membrane (CAM) assay. T-HEp3 cells are inoculated onto the CAM with type I, III, or IV collagen or with Dulbecco’s phosphate-buffered saline (DPBS) as a vehicle control. T-HEp3 cells co-injected with recombinant collagen fragments and the vehicle control are observed for tumor cell growth. T-HEp3 cells are injected into the right flank and T-HEp3 cells are injected contralaterally in nude mice with type III collagen. Tumor growth is observed.

[0325] 4T1 or D2A1 cells are orthotopically injected into mice and recombinant collagen fragments are co-injected. Phospho-histone H3 protein levels are observed to determine cell proliferation. Nuclear quiescence markers are observed and live cell imaging of T-HEp3 cells expressing a CDK2 cell cycle sensor is performed to determine induction of quiescence.

[0326] It should be understood that the section "Mode for Carrying Out the Invention" is intended to be used for interpreting the scope of the claims, rather than the sections "Summary of the Invention" and "Abstract". The sections "Summary of the Invention" and "Abstract" may describe one or more but not all exemplary embodiments of the present invention as contemplated by the inventor, but are never intended to limit the present invention and the scope of the appended claims.

Claims

1. A recombinant collagen fragment used in a method for treating cancer, wherein the recombinant collagen fragment has at least about 85% sequence identity with the amino acid sequence described in SEQ ID NO: 1, or has the amino acid sequence described in any one of SEQ ID NOs: 2 to 972, SEQ ID NO: 973, or SEQ ID NOs: 974 to 1002.

2. The recombinant collagen fragment according to claim 1, wherein the recombinant collagen fragment has the amino acid sequence described in SEQ ID NO:

1.

3. The recombinant collagen fragment according to claim 1, wherein the cancer is characterized by the presence of a solid tumor.

4. The recombinant collagen fragment according to claim 1, wherein the recombinant collagen fragment is formulated in a pharmaceutically acceptable composition.

5. The recombinant collagen fragment according to claim 1, wherein the recombinant collagen fragment is formulated in a therapeutic biomaterial.

6. The recombinant collagen fragment according to claim 5, wherein the therapeutic biomaterial is a protein polyurethane alloy.

7. The recombinant collagen fragment according to claim 6, wherein the protein is dissolved in the polyurethane.

8. The recombinant collagen fragment according to claim 6 or 7, wherein the alloy comprises about 10% to about 50% by weight of the fragment and about 50% to about 90% by weight of the polyurethane.

9. The recombinant collagen fragment according to claim 6 or 7, wherein the alloy comprises about 20% to about 35% by weight of the protein and about 65% to about 80% by weight of the polyurethane.

10. The recombinant collagen fragment according to any one of claims 6 or 7, wherein the alloy does not contain or substantially contains particles of the recombinant collagen fragment having an average diameter of more than 1 micron.

11. A therapeutic biomaterial comprising a recombinant collagen fragment having at least about 85% sequence identity with the amino acid sequence described in SEQ ID NO: 1, SEQ ID NOs: 2-972, SEQ ID NO: 973, or SEQ ID NOs: 974-1002, and a pharmaceutically acceptable scaffold.

12. The therapeutic biomaterial according to claim 11, wherein the collagen fragment has the amino acid sequence described in Sequence ID No.

1.

13. The therapeutic biomaterial according to claim 11, wherein the pharmaceutically acceptable scaffold comprises (i) a mixture of hyaluronic acid, polyvinylpyrrolidone, polyacrylamide, poly(ethylene oxide), poly(2-oxazoline), polyethyleneimine, carboxymethylcellulose, chondroitin sulfate, acetylated hyaluronate, or zinc hyaluronate and (ii) the recombinant collagen fragment.

14. A therapeutic biomaterial according to any one of claims 11 to 13, used in a method for treating wounds resulting from the excision of a primary tumor.

Images